JP7339723B2 - Mutant cytochrome protein and its use - Google Patents

Mutant cytochrome protein and its use Download PDF

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JP7339723B2
JP7339723B2 JP2017131345A JP2017131345A JP7339723B2 JP 7339723 B2 JP7339723 B2 JP 7339723B2 JP 2017131345 A JP2017131345 A JP 2017131345A JP 2017131345 A JP2017131345 A JP 2017131345A JP 7339723 B2 JP7339723 B2 JP 7339723B2
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cytochrome
burkholderia
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JP2019013166A (en
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広司 早出
順子 島▲崎▼
一茂 森
勝博 小嶋
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Arkray Inc
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    • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
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Description

本発明は低電位でも電子授受が可能な変異型シトクロムタンパク質に関する。本発明の変異型シトクロムタンパク質はバイオセンサ等に好適に使用することができ、生化学分野、医療分野等で有用である。 TECHNICAL FIELD The present invention relates to mutant cytochrome proteins capable of electron transfer even at low potential. The mutant cytochrome protein of the present invention can be suitably used for biosensors and the like, and is useful in the fields of biochemistry, medicine, and the like.

電子受容タンパク質としてはシトクロムタンパク質が知られており、酸化還元酵素の触媒サブユニットと複合体を形成させて電気化学的センサとして使用されている。
例えば、特許文献１には、ブルクホルデリア属の微生物に由来する、グルコースデヒドロゲナーゼの触媒サブユニット（αサブユニット）とシトクロムＣサブユニット（βサブユニット）の複合体をバイオセンサとして使用することが開示されている（特許文献１）。このようなバイオセンサは生体試料中のグルコース濃度などの測定に使用されるが、より精度よく測定するためにはタンパク質を改変するなどにより、測定感度を向上させることが求められる。
これまでに、電気化学的センサタンパク質の変異体としては主に触媒サブユニットの変異体が研究されており、シトクロムタンパク質の変異体はほとんど研究されてこなかった。 Cytochrome proteins are known as electron-accepting proteins, and are used as electrochemical sensors by forming complexes with catalytic subunits of oxidoreductases.
For example, Patent Document 1 discloses that a complex of a catalytic subunit (α subunit) of glucose dehydrogenase and a cytochrome C subunit (β subunit) derived from a microorganism belonging to the genus Burkholderia can be used as a biosensor. It is disclosed (Patent Document 1). Such biosensors are used to measure the glucose concentration in biological samples, but in order to measure them more accurately, it is required to improve the measurement sensitivity by modifying the protein.
So far, mutants of electrochemical sensor proteins have mainly been studied in the catalytic subunit, and few in cytochrome proteins.

WO 2005/023111WO 2005/023111

酸化還元電流を観測するバイオセンサを用いた目的物質の測定においては、試料中に含まれる還元物質の影響を低減するために、酵素および酵素複合体と電極間の電子授受が観測される範囲で酸化還元電位を下げることが望ましい。 In the measurement of the target substance using a biosensor that observes redox current, in order to reduce the influence of reducing substances contained in the sample, the electron transfer between the enzyme or enzyme complex and the electrode is observed. It is desirable to lower the redox potential.

しかしながら、従来の天然のシトクロムタンパク質を電子受容タンパク質として用いて酸化還元電流を観測する場合には電極に大きな電位の印加を要する。例えば、特許文献１の実施例７では銀・塩化銀電極に対して＋３５０ｍＶの電位を印加しており、この電位においてはアスコルビン酸やアセトアミノフェンのような還元性物質が酸化されることによる測定値への正バイアスが生じることが懸念される。 However, when conventional natural cytochrome proteins are used as electron-accepting proteins and redox currents are observed, a large potential must be applied to the electrodes. For example, in Example 7 of Patent Document 1, a potential of +350 mV is applied to the silver/silver chloride electrode, and at this potential, reducing substances such as ascorbic acid and acetaminophen are oxidized. We are concerned about the positive bias to the value.

このような問題点を鑑み、本発明は、低電位印加で目的物質の測定が可能なバイオセンサに利用可能な変異型電子受容サブユニットタンパク質を提供することを課題とする。 In view of such problems, an object of the present invention is to provide a mutant electron acceptor subunit protein that can be used for a biosensor that can measure a target substance with low potential application.

本発明者は、上記課題を解決するために、アミノ末端から数えて１番目の第１ヘム結合領域および２番目の第２ヘム結合領域を含む領域を欠損させた変異型シトクロムＣタンパク質を作製し、これを用いてバイオセンサの評価を行った。その結果、驚くべきことに、第１および第２ヘム結合領域を含む領域を欠損させた変異型シトクロムＣタンパク質はヘム結合領域が１つ（第３のヘム結合領域）しかないにもかかわらず触媒サブユニットから電子を受容することができ、従来よりも低電位で目的物質に応じた電流を流すことができることを見出し、本発明を完成するに至った。 In order to solve the above problems, the present inventors prepared a mutant cytochrome C protein in which a region containing the first heme-binding region and the second heme-binding region counted from the amino terminus is deleted. , which was used to evaluate biosensors. As a result, surprisingly, the mutant cytochrome C protein lacking the region containing the first and second heme-binding regions has only one heme-binding region (the third heme-binding region), but the catalytic The inventors have found that electrons can be accepted from subunits, and that a current corresponding to a target substance can flow at a lower potential than in the past, leading to the completion of the present invention.

すなわち、本発明は以下のとおりである。
［１］３つのヘム結合ドメインを有するシトクロムタンパク質において、Ｎ末端側から数
えて１番目の第１ヘム結合ドメインおよび２番目の第２のヘム結合ドメインが欠損した、変異型シトクロムタンパク質。
［２］前記第１および第２のヘム結合ドメインを含む領域が欠損した、［１］に記載の変異型シトクロムタンパク質。
［３］前記第１および第２のヘム結合ドメインを含む領域が配列番号４の４３～１９５の領域に相当する、［２］に記載の変異型シトクロムタンパク質。
［４］シトクロムタンパク質がシトクロムＣタンパク質である、［１］～［３］のいずれかに記載の変異型シトクロムタンパク質。
［５］シトクロムＣタンパク質がブルクホルデリア属微生物に由来する、［４］に記載の変異型シトクロムタンパク質。
［６］シトクロムＣタンパク質がブルクホルデリア・セパシアに由来する、［５］に記載の変異型シトクロムタンパク質。
［７］改変前のシトクロムタンパク質が、配列番号４と６０％以上のアミノ酸配列同一性を有する、［１］～［６］のいずれかに記載の変異型シトクロムタンパク質。
［８］配列番号４のアミノ酸番号３１４～４２５のアミノ酸配列、配列番号４のアミノ酸番号３３０～４２５のアミノ酸配列、またはこれらのアミノ酸配列において１または数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列（ただし、アミノ酸番号３３４～３３８のＣＸＸＣＨモチーフは改変されない）からなる、［１］～［７］のいずれかに記載の変異型シトクロムタンパク質。
［９］［１］～［８］のいずれかに記載の変異型シトクロムタンパク質をコードするＤＮＡ。
［１０］［９］に記載のＤＮＡを含む組み換えベクター。
［１１］［１０］に記載の組み換えベクターで形質転換された形質転換細胞。
［１２］［１］～［８］のいずれかに記載の変異型シトクロムタンパク質および酸化還元酵素の触媒サブユニットタンパク質を含む、酸化還元酵素・シトクロム複合体。
［１３］酸化還元酵素がグルコースデヒドロゲナーゼである、［１２］に記載の酸化還元酵素・シトクロム複合体。
［１４］［１２］または［１３］に記載の酸化還元酵素・シトクロム複合体を含むバイオセンサ。
［１５］［１４］に記載のバイオセンサに試料を添加し、電位を印加して応答電流を測定し、応答電流に基づいて試料に含まれる測定対象物質の濃度を算出することを特徴とする、測定対象物質の測定方法。
［１６］印加電位が銀・塩化銀電極に対して０～＋３００ｍＶである、［１５］に記載の測定方法。 That is, the present invention is as follows.
[1] A mutant cytochrome protein in which the first heme-binding domain and the second heme-binding domain counted from the N-terminal side of the cytochrome protein having three heme-binding domains are deleted.
[2] The mutant cytochrome protein of [1], wherein the region containing the first and second heme-binding domains is deleted.
[3] The mutant cytochrome protein of [2], wherein the region containing the first and second heme-binding domains corresponds to the region 43-195 of SEQ ID NO:4.
[4] The mutant cytochrome protein according to any one of [1] to [3], wherein the cytochrome protein is cytochrome C protein.
[5] The mutant cytochrome protein according to [4], wherein the cytochrome C protein is derived from a Burkholderia microorganism.
[6] The mutant cytochrome protein of [5], wherein the cytochrome C protein is derived from Burkholderia cepacia.
[7] The mutant cytochrome protein according to any one of [1] to [6], wherein the cytochrome protein before modification has 60% or more amino acid sequence identity with SEQ ID NO:4.
[8] the amino acid sequence of amino acid numbers 314 to 425 of SEQ ID NO: 4, the amino acid sequence of amino acid numbers 330 to 425 of SEQ ID NO: 4, or substitution, deletion, insertion or addition of one or several amino acids in these amino acid sequences The mutant cytochrome protein according to any one of [1] to [7], which consists of a modified amino acid sequence (wherein the CXXCH motifs of amino acid numbers 334 to 338 are not modified).
[9] A DNA encoding the mutant cytochrome protein according to any one of [1] to [8].
[10] A recombinant vector containing the DNA of [9].
[11] A transformed cell transformed with the recombinant vector of [10].
[12] An oxidoreductase/cytochrome complex comprising the mutant cytochrome protein of any one of [1] to [8] and an oxidoreductase catalytic subunit protein.
[13] The oxidoreductase-cytochrome complex of [12], wherein the oxidoreductase is glucose dehydrogenase.
[14] A biosensor comprising the oxidoreductase/cytochrome complex of [12] or [13].
[15] A sample is added to the biosensor according to [14], a potential is applied to measure a response current, and the concentration of the substance to be measured contained in the sample is calculated based on the response current. , a method for measuring a substance to be measured.
[16] The measuring method according to [15], wherein the applied potential is 0 to +300 mV with respect to the silver/silver chloride electrode.

本発明によれば、従来より低い電位（例えば、銀・塩化銀電極に対して０～＋３００ｍＶ、好ましくは０～＋１５０ｍＶ、より好ましくは０～＋１００ｍＶ）で作動する電気化学的バイオセンサの構築が可能であり、試料中の還元物質による干渉を抑えて、グルコース等の測定対象物質の正確な測定をすることが可能となる。 According to the present invention, it is possible to construct an electrochemical biosensor that operates at a potential lower than conventional (for example, 0 to +300 mV, preferably 0 to +150 mV, more preferably 0 to +100 mV against silver/silver chloride electrode). , it is possible to accurately measure a substance to be measured such as glucose by suppressing interference due to reducing substances in the sample.

野生型βサブユニットを含むグルコースデヒドロゲナーゼ（ＧＤＨ）複合体を含むグルコースセンサ、またはＮ末側欠損型βサブユニットを含むＧＤＨ複合体を含むグルコースセンサを用いたクロノアンペアメトリーの結果を示す図（印加電位は０，＋１００，＋４００ｍＶ）。A diagram showing the results of chronoamperemetry using a glucose sensor containing a glucose dehydrogenase (GDH) complex containing a wild-type β subunit or a glucose sensor containing a GDH complex containing an N-terminal truncated β subunit (applied potentials are 0, +100, +400 mV).

以下、本発明を詳細に説明する。
本発明の変異型シトクロムタンパク質は、３つのヘム結合ドメインを有するシトクロムタ
ンパク質の変異体であって、Ｎ末端側から数えて１番目の第１ヘム結合ドメインおよび２番目の第２のヘム結合ドメインが欠損した、変異型シトクロムタンパク質である。 The present invention will be described in detail below.
The mutant cytochrome protein of the present invention is a cytochrome protein mutant having three heme-binding domains, wherein the first heme-binding domain and the second heme-binding domain counted from the N-terminal side are A defective, mutant cytochrome protein.

シトクロムタンパク質はヘム結合ドメインを３つ有しており、本明細書では、Ｎ末側から数えて１，２，３番目のヘム結合ドメインをそれぞれ第１、第２、第３のヘム結合ドメインと呼ぶ。ヘム結合ドメインは一般的にＣＸＸＣＨ（配列番号５）（Ｘは任意のアミノ酸）で表され、本発明の変異型シトクロムタンパク質では第１および第２のヘム結合ドメインが欠損するように改変されている。 A cytochrome protein has three heme-binding domains, and in this specification, the 1st, 2nd and 3rd heme-binding domains counted from the N-terminus are referred to as the 1st, 2nd and 3rd heme-binding domains, respectively. call. The heme-binding domain is generally represented by CXXCH (SEQ ID NO: 5) (X is any amino acid), and the mutant cytochrome proteins of the present invention are modified to lack the first and second heme-binding domains. .

ここで、「第１および第２のヘム結合ドメインが欠損するように改変されている」とは、第１および第２のＣＸＸＣＨモチーフが欠損されていることを意味する。 Here, "modified to lack the first and second heme-binding domains" means lacking the first and second CXXCH motifs.

ＣＸＸＣＨモチーフの欠損には、ＣＸＸＣＨモチーフを含む領域の欠損も含まれ、第１ヘム結合ドメインを含む領域と第２ヘム結合ドメインを含む領域のそれぞれが欠損していてもよいし、第１および第２ヘム結合ドメインを含む領域が欠損していてもよい。 Deletion of the CXXCH motif also includes deletion of the region containing the CXXCH motif, and the region containing the first heme-binding domain and the region containing the second heme-binding domain may be deleted, or The region containing the two heme-binding domains may be deleted.

シトクロムタンパク質としては、シトクロムＣタンパク質が好ましく、配列番号４で表されるブルクホリデリア・セパシアのシトクロムＣタンパク質（グルコースデヒドロゲナーゼのβサブユニット）が挙げられる。
以下、ブルクホリデリア・セパシアのシトクロムＣタンパク質を代表例として説明する。 The cytochrome protein is preferably a cytochrome C protein, and includes the cytochrome C protein of Burchholideria cepacia represented by SEQ ID NO: 4 (β subunit of glucose dehydrogenase).
The cytochrome C protein of Burchholideria cepacia will be described below as a representative example.

配列番号４においては、第１のヘム結合ドメイン（アミノ酸番号４３～４７）、第２のヘム結合ドメイン（アミノ酸番号１９１～１９５）、第３のヘム結合ドメイン（アミノ酸番号３３４～３３８）が存在する。これら第１ヘム結合ドメインおよび第２ヘム結合ドメインが欠損されるが、第１および第２ヘム結合ドメインを含む領域（アミノ酸番号４３～１９５）が欠損していてもよい。 In SEQ ID NO: 4, there is a first heme-binding domain (amino acid numbers 43-47), a second heme-binding domain (amino acid numbers 191-195), and a third heme-binding domain (amino acid numbers 334-338). . Although the first and second heme-binding domains are deleted, the region containing the first and second heme-binding domains (amino acid numbers 43-195) may be deleted.

第３のヘム結合ドメインが維持され、変異型シトクロムＣタンパク質としての機能を発揮する限り、すなわち、電子授受機能が損なわれない限り、第１および第２ヘム結合ドメインを含む領域（アミノ酸番号４３～１９５）は少なくともこの領域が欠損していればよく、配列番号４におけるその前後のアミノ酸を含めて欠損してもよい。例えば、配列番号４のＮ末側からアミノ酸番号１９５までが欠損してもよく、例えば、第１および第２ヘム結合ドメインを含む領域が欠損した変異型シトクロムＣタンパク質として配列番号４のアミノ酸番号３１４～４２５または３３０～４２５からなる変異型シトクロムＣタンパク質が挙げられる。ただし、これらの配列において、Ｎ末端側及び／またはＣ末端側に任意のタグ配列やシグナル配列（例えば、配列番号４のアミノ酸番号１～２７）などが付加されてもよい。 The region containing the first and second heme-binding domains (amino acid number 43 to 195) may lack at least this region, and may also lack amino acids before and after it in SEQ ID NO:4. For example, amino acid number 195 from the N-terminal side of SEQ ID NO: 4 may be deleted. ~425 or mutant cytochrome C proteins consisting of 330-425. However, in these sequences, any tag sequence or signal sequence (eg, amino acid numbers 1 to 27 of SEQ ID NO:4) may be added to the N-terminal side and/or C-terminal side.

なお、本発明の変異型シトクロムタンパク質は配列番号４のアミノ酸番号３１４～４２５からなるアミノ酸配列または配列番号４のアミノ酸番号３３０～４２５からなるアミノ酸配列そのものに限定されず、電子授受機能が損なわれない限り、これらの配列において、第３のヘム結合ドメイン（アミノ酸番号３３４～３３８）以外の１または数個のアミノ酸が置換、欠失、付加または挿入された配列でもよい。ここで、「１又は数個」とは、例えば、１～２０個、好ましくは１～１０個、より好ましくは１～８個、さらに好ましくは１～５個、特に好ましくは１～３個である。また、置換は保存的な置換が好ましく、「保存的置換」とは、酸性アミノ酸同士の置換、中性アミノ酸同士の置換、塩基性アミノ酸同士の置換など、性質が類似したアミノ酸同士の置換をいう。また、配列番号４のアミノ酸番号３１４～４２５からなるアミノ酸配列のＮ末側には、配列番号４のアミノ酸番号１９６～３１３からなるアミノ酸配列のＣ末側配列の一部が付加されてよいし、配列番号４のアミノ酸番号３３０～４２５からなるアミノ酸配列のＮ末側には、配列番号４のアミノ酸番
号１９６～３２９からなるアミノ酸配列のＣ末側配列の一部が付加されてよい。
なお、本発明の変異型シトクロムＣタンパク質は、第３のヘム結合ドメイン（アミノ酸番号３３４～３３８）が保持され、電子授受機能が損なわれない限り、配列番号４のアミノ酸番号３１４～４２５からなるアミノ酸配列または配列番号４の３３０～４２５からなるアミノ酸配列と、８０％以上、好ましくは９０％以上、より好ましくは９５％以上の配列同一性を有するアミノ酸配列を有するタンパク質であってもよい。 In addition, the mutant cytochrome protein of the present invention is not limited to the amino acid sequence consisting of amino acid numbers 314 to 425 of SEQ ID NO: 4 or the amino acid sequence itself consisting of amino acid numbers 330 to 425 of SEQ ID NO: 4, and the electron transfer function is not impaired. As long as these sequences have one or several amino acids other than the third heme-binding domain (amino acid numbers 334 to 338) substituted, deleted, added or inserted. Here, "one or several" means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 5, particularly preferably 1 to 3. be. In addition, substitutions are preferably conservative substitutions, and "conservative substitutions" refer to substitutions between amino acids with similar properties, such as substitutions between acidic amino acids, substitutions between neutral amino acids, and substitutions between basic amino acids. . In addition, a part of the C-terminal sequence of the amino acid sequence consisting of amino acid numbers 196 to 313 of SEQ ID NO: 4 may be added to the N-terminal side of the amino acid sequence consisting of amino acid numbers 314 to 425 of SEQ ID NO: 4, A part of the C-terminal side sequence of the amino acid sequence consisting of amino acid numbers 196-329 of SEQ ID NO:4 may be added to the N-terminal side of the amino acid sequence consisting of amino acid numbers 330-425 of SEQ ID NO:4.
The mutant cytochrome C protein of the present invention retains the third heme-binding domain (amino acid numbers 334 to 338) and does not impair the electron transfer function. It may be a protein having an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more sequence identity with the sequence or the amino acid sequence consisting of 330-425 of SEQ ID NO:4.

なお、配列番号４はブルクホリデリア・セパシアＫＳ１株が保持するＧＤＨ βサブユニットのアミノ酸配列であるが、ＫＳ１株は、平成１２年９月２５日に独立行政法人産業技術総合研究所特許生物寄託センター（現独立行政法人製品評価技術基盤機構(ＮＩＴＥ)〒292-0818 日本国千葉県木更津市かずさ鎌足２－５－８）に、受託番号第ＦＥＲＭ ＢＰ－７３０６として寄託されている。 SEQ ID NO: 4 is the amino acid sequence of the GDH β subunit retained by the Burkholideria cepacia strain KS1. It has been deposited with the Center (now National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) under the accession number FERM BP-7306.

本発明において、上記改変が導入される前のシトクロムＣタンパク質は配列番号４で表されるブルクホリデリア・セパシアのシトクロムＣタンパク質に限定されず、第１、第２、第３のヘム結合ドメインを有するものであればよいが、例えば、配列番号４のホモログが使用でき、好ましくは配列番号４と６０％以上、好ましくは８０％以上、より好ましくは９０%以上の同一性を有する配列番号４のホモログが使用できる。 In the present invention, the cytochrome C protein before the introduction of the above modifications is not limited to the cytochrome C protein of Burchholideria cepacia represented by SEQ ID NO: 4, and the first, second, and third heme-binding domains are For example, a homologue of SEQ ID NO: 4 can be used, and preferably has 60% or more, preferably 80% or more, more preferably 90% or more identity with SEQ ID NO: 4. Homologs are available.

配列番号４と６０％以上の配列同一性を有するシトクロムＣタンパク質のアミノ酸配列としては、ブルクホリデリア属のシトクロムＣタンパク質が好ましく、Burkholderia cepacia J2315株のシトクロムタンパク質（配列番号６）、Burkholderia cenocepaciaのシトクロムＣタンパク質（配列番号７）、Burkholderia multivoransのシトクロムＣタンパク質（配列番号８）、Burkholderia ubonensisのシトクロムＣタンパク質（配列番号９）、Burkholderia stagnalisのシトクロムＣタンパク質（配列番号１０）、Burkholderia thailandensisのシトクロムＣタンパク質（配列番号１１）が例示される。
また、ブルクホリデリア属以外のシトクロムタンパク質であってもよく、例えば、Ralstonia solanacearumのシトクロムタンパク質（配列番号１２）やRalstonia pickettiiのシ
トクロムタンパク質（配列番号１３）が例示される。ただし、これらに限定はされず、その他の生物由来のシトクロムＣタンパク質でもよい。配列番号４と６０％以上の配列同一性を有するシトクロムＣタンパク質のアミノ酸配列の例をNational Center for Biotechnology Information（NCBI）のProtein Databaseのアクセション番号にて以下に示す。こ
れらの配列ではヘム結合ドメインが３つ存在し、アミノ末端側から数えて１、２番目の第１ヘム結合ドメインおよび第２ヘム結合ドメインを欠損させることで本発明の変異型シトクロムタンパク質が得られる。 The amino acid sequence of the cytochrome C protein having a sequence identity of 60% or more with SEQ ID NO: 4 is preferably the cytochrome C protein of the genus Burkholderia, the cytochrome protein of the Burkholderia cepacia J2315 strain (SEQ ID NO: 6), and the cytochrome of Burkholderia cenocepacia. C protein (SEQ ID NO: 7), Burkholderia multivorans cytochrome C protein (SEQ ID NO: 8), Burkholderia ubonensis cytochrome C protein (SEQ ID NO: 9), Burkholderia stagnalis cytochrome C protein (SEQ ID NO: 10), Burkholderia thailandensis cytochrome C protein (SEQ ID NO: 11) is exemplified.
Cytochrome proteins other than those belonging to the genus Burkholideria may also be used, for example, the cytochrome protein of Ralstonia solanacearum (SEQ ID NO: 12) and the cytochrome protein of Ralstonia pickettii (SEQ ID NO: 13). However, it is not limited to these, and cytochrome C proteins derived from other organisms may be used. Examples of amino acid sequences of cytochrome C proteins having 60% or more sequence identity with SEQ ID NO: 4 are shown below under the accession numbers of the Protein Database of the National Center for Biotechnology Information (NCBI). These sequences have three heme-binding domains, and the mutant cytochrome protein of the present invention can be obtained by deleting the first and second heme-binding domains counted from the amino terminal side. .

WP_006403391.1 MULTISPECIES: cytochrome C [Burkholderia]
AAQ06608.1 glucose dehydrogenase beta subunit [Burkholderia cepacia]
WP_006396899.1 cytochrome C [Burkholderia multivorans]（配列番号８）
SAJ95286.1 gluconate 2-dehydrogenase (acceptor) [Burkholderia multivorans]
WP_006412653.1 cytochrome C [Burkholderia multivorans]
WP_060041792.1 cytochrome C [Burkholderia multivorans]
WP_060112921.1 cytochrome C [Burkholderia multivorans]
WP_060182288.1 cytochrome C [Burkholderia multivorans]
WP_060151834.1 cytochrome C [Burkholderia multivorans]
WP_048804658.1 cytochrome C [Burkholderia multivorans]
WP_059786407.1 cytochrome C [Burkholderia multivorans]
WP_035955019.1 cytochrome C [Burkholderia multivorans]
WP_059585013.1 cytochrome C [Burkholderia anthina]
WP_006482958.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]（配列番
号６）
WP_048988065.1 cytochrome C [Burkholderia cenocepacia] （配列番号７）
WP_084751507.1 cytochrome C [Burkholderia cenocepacia]
WP_077186512.1 cytochrome C [Burkholderia cenocepacia]
WP_069351905.1 cytochrome C [Burkholderia cenocepacia]
WP_059783319.1 cytochrome C [Burkholderia sp. NRF60-BP8]
WP_060263394.1 cytochrome C [Burkholderia cenocepacia]
WP_059836247.1 cytochrome C [Burkholderia sp. MSMB1835]
WP_059556637.1 cytochrome C [Burkholderia seminalis]
WP_063551853.1 cytochrome C [Burkholderia territorii]
WP_060310848.1 cytochrome C [Burkholderia anthina]
WP_034187893.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_059789576.1 cytochrome C [Burkholderia sp. MSMB1072]
WP_059500343.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_034204695.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_011882359.1 cytochrome C [Burkholderia vietnamiensis]
WP_060968838.1 cytochrome C [Burkholderia anthina]
WP_077204198.1 cytochrome C [Burkholderia cenocepacia]
WP_050012790.1 cytochrome C [Burkholderia cenocepacia]
WP_011547563.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_014724780.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_059453668.1 cytochrome C [Burkholderia vietnamiensis]
WP_034195126.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_027812584.1 cytochrome C [Burkholderia cenocepacia]
WP_059720348.1 cytochrome C [Burkholderia vietnamiensis]
WP_059548127.1 cytochrome C [Burkholderia vietnamiensis]
WP_044843288.1 cytochrome C [Burkholderia sp. USM B20]
WP_059577619.1 cytochrome C [Burkholderia vietnamiensis]
WP_059459380.1 cytochrome C [Burkholderia vietnamiensis]
WP_059451171.1 cytochrome C [Burkholderia territorii]
WP_027780832.1 cytochrome C [Burkholderia cepacia]
WP_077217239.1 cytochrome C [Burkholderia cenocepacia]
WP_059539736.1 cytochrome C [Burkholderia diffusa]
WP_059507572.1 cytochrome C [Burkholderia territorii]
AOJ19136.1 cytochrome C [Burkholderia cenocepacia]
WP_027806162.1 cytochrome C [Burkholderia cenocepacia]
WP_060107812.1 cytochrome C [Burkholderia territorii]
WP_059822261.1 cytochrome C [Burkholderia sp. MSMB1826]
WP_059464697.1 cytochrome C [Burkholderia diffusa]
WP_059240315.1 cytochrome C [Burkholderia cepacia]
WP_059734410.1 cytochrome C [Burkholderia vietnamiensis]
WP_069227164.1 cytochrome C [Burkholderia diffusa]
WP_060118353.1 cytochrome C [Burkholderia territorii]
WP_069617959.1 cytochrome C [Burkholderia sp. A2]
WP_060347293.1 cytochrome C [Burkholderia territorii]
WP_059702517.1 cytochrome C [Burkholderia vietnamiensis]
WP_060126789.1 cytochrome C [Burkholderia territorii]
WP_069260162.1 cytochrome C [Burkholderia metallica]
WP_059978049.1 cytochrome C [Burkholderia territorii]
WP_059691288.1 cytochrome C [Burkholderia sp. RF4-BP95]
WP_040140035.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_011349243.1 cytochrome C [Burkholderia lata]
WP_059533875.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_059547167.1 cytochrome C [Burkholderia latens]
WP_057924955.1 cytochrome C [Burkholderia ambifaria]
WP_059607236.1 cytochrome C [Burkholderia sp. LA-2-3-30-S1-D2]
WP_039351157.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_046548033.1 cytochrome C [Burkholderia contaminans]
WP_006752013.1 cytochrome C [Burkholderia ambifaria]
WP_014899067.1 cytochrome C [Burkholderia cepacia]
WP_006756339.1 cytochrome C [Burkholderia ambifaria]
WP_059819259.1 cytochrome C [Burkholderia sp. MSMB0856]
WP_011354899.1 cytochrome C [Burkholderia lata]
WP_039320755.1 cytochrome C [Burkholderia sp. A9]
WP_035974222.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_071332428.1 cytochrome C [Burkholderia contaminans]
WP_059684703.1 cytochrome C [Burkholderia sp. FL-7-2-10-S1-D7]
WP_048252126.1 cytochrome C [Burkholderia cepacia]
WP_069251790.1 cytochrome C [Burkholderia lata]
WP_011658978.1 cytochrome C [Burkholderia ambifaria]
WP_012366204.1 cytochrome C [Burkholderia ambifaria]
WP_072438780.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_031400525.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_034180248.1 cytochrome C [Burkholderia pyrrocinia]
WP_043184377.1 cytochrome C [Burkholderia cepacia]
WP_059729924.1 cytochrome C [Burkholderia cepacia]
WP_059713002.1 cytochrome C [Burkholderia ubonensis]（配列番号９）
WP_065501791.1 cytochrome C [Burkholderia stabilis]
WP_059615074.1 cytochrome C [Burkholderia ubonensis]
WP_085037374.1 cytochrome C [Burkholderia sp. CAMPA 1040]
WP_060359188.1 cytochrome C [Burkholderia cepacia]
WP_060333590.1 cytochrome C [Burkholderia ubonensis]
WP_059522548.1 cytochrome C [Burkholderia cepacia]
WP_059766841.1 cytochrome C [Burkholderia ubonensis]
WP_059637760.1 cytochrome C [Burkholderia ubonensis]
WP_040131620.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_060052348.1 cytochrome C [Burkholderia cepacia]
WP_021162032.1 MULTISPECIES: glucose dehydrogenase [Burkholderia]
WP_059666051.1 cytochrome C [Burkholderia cepacia]
WP_060375463.1 cytochrome C [Burkholderia cepacia]
WP_060371861.1 cytochrome C [Burkholderia cepacia]
WP_059697397.1 cytochrome C [Burkholderia cepacia]
WP_047903070.1 cytochrome C [Burkholderia pyrrocinia]
WP_027791850.1 cytochrome C [Burkholderia cepacia]
WP_059552416.1 cytochrome C [Burkholderia ubonensis]
WP_060232513.1 cytochrome C [Burkholderia cepacia]
WP_059687063.1 cytochrome C [Burkholderia cepacia]
WP_060226065.1 cytochrome C [Burkholderia cepacia]
WP_060125713.1 cytochrome C [Burkholderia cepacia]
WP_059923490.1 cytochrome C [Burkholderia stagnalis]（配列番号１０）
WP_059565670.1 cytochrome C [Burkholderia stagnalis]
OJD09200.1 cytochrome C [Burkholderia sp. DNA89]
WP_060237338.1 cytochrome C [Burkholderia pseudomultivorans]
WP_059707234.1 cytochrome C [Burkholderia cepacia]
WP_060362538.1 cytochrome C [Burkholderia stagnalis]
WP_060159353.1 cytochrome C [Burkholderia stagnalis]
WP_069748432.1 cytochrome C [Burkholderia stabilis]
WP_059677336.1 cytochrome C [Burkholderia cepacia]
WP_059730424.1 cytochrome C [Burkholderia ubonensis]
WP_059813883.1 cytochrome C [Burkholderia cepacia]
WP_059993161.1 cytochrome C [Burkholderia stagnalis]
WP_060236459.1 cytochrome C [Burkholderia ubonensis]
WP_076476708.1 cytochrome C [Burkholderia ubonensis]
WP_010089725.1 cytochrome C [Burkholderia ubonensis]
WP_060195541.1 cytochrome C [Burkholderia ubonensis]
WP_059971786.1 cytochrome C [Burkholderia pyrrocinia]
WP_060367052.1 cytochrome C [Burkholderia ubonensis]
WP_060016611.1 cytochrome C [Burkholderia ubonensis]
WP_059924260.1 cytochrome C [Burkholderia ubonensis]
WP_060048488.1 cytochrome C [Burkholderia ubonensis]
WP_059892434.1 cytochrome C [Burkholderia ubonensis]
WP_059488090.1 cytochrome C [Burkholderia ubonensis]
WP_059483872.1 cytochrome C [Burkholderia ubonensis]
WP_059865488.1 cytochrome C [Burkholderia ubonensis]
WP_059633519.1 cytochrome C [Burkholderia ubonensis]
WP_060287380.1 cytochrome C [Burkholderia ubonensis]
WP_059853060.1 cytochrome C [Burkholderia ubonensis]
WP_059968663.1 cytochrome C [Burkholderia ubonensis]
WP_059752176.1 cytochrome C [Burkholderia ubonensis]
WP_059532869.1 cytochrome C [Burkholderia ubonensis]
WP_059660535.1 cytochrome C [Burkholderia ubonensis]
WP_059590294.1 cytochrome C [Burkholderia ubonensis]
WP_026043943.1 cytochrome C [Burkholderia pyrrocinia]
WP_060264220.1 cytochrome C [Burkholderia ubonensis]
WP_060019510.1 cytochrome C [Burkholderia ubonensis]
WP_059946330.1 cytochrome C [Burkholderia ubonensis]
WP_042588017.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_084908871.1 cytochrome C [[Pseudomonas] mesoacidophila]
WP_071751088.1 cytochrome C [Burkholderia ubonensis]
WP_060094261.1 cytochrome C [Burkholderia ubonensis]
WP_059885808.1 cytochrome C [Burkholderia ubonensis]
WP_059878406.1 cytochrome C [Burkholderia ubonensis]
WP_071852856.1 cytochrome C [Burkholderia ubonensis]
WP_060161154.1 cytochrome C [Burkholderia ubonensis]
WP_059796027.1 cytochrome C [Burkholderia ubonensis]
WP_059835010.1 cytochrome C [Burkholderia ubonensis]
WP_059911385.1 cytochrome C [Burkholderia ubonensis]
WP_059744627.1 cytochrome C [Burkholderia ubonensis]
WP_045565069.1 cytochrome C [Burkholderia ubonensis]
WP_060276465.1 cytochrome C [Burkholderia ubonensis]
WP_060052180.1 cytochrome C [Burkholderia ubonensis]
WP_059877204.1 cytochrome C [Burkholderia ubonensis]
WP_059918860.1 cytochrome C [Burkholderia ubonensis]
WP_059674217.1 cytochrome C [Burkholderia ubonensis]
WP_059626644.1 cytochrome C [Burkholderia ubonensis]
WP_059706954.1 cytochrome C [Burkholderia ubonensis]
WP_059610359.1 cytochrome C [Burkholderia ubonensis]
WP_071751781.1 cytochrome C [Burkholderia ubonensis]
WP_060345076.1 cytochrome C [Burkholderia ubonensis]
WP_060056668.1 cytochrome C [Burkholderia ubonensis]
WP_059867004.1 cytochrome C [Burkholderia ubonensis]
WP_059846198.1 cytochrome C [Burkholderia ubonensis]
WP_059956839.1 cytochrome C [Burkholderia ubonensis]
WP_059801215.1 cytochrome C [Burkholderia ubonensis]
WP_060374144.1 cytochrome C [Burkholderia ubonensis]
WP_060228155.1 cytochrome C [Burkholderia ubonensis]
WP_060449283.1 cytochrome C [Burkholderia ubonensis]
WP_060088315.1 cytochrome C [Burkholderia ubonensis]
WP_060141594.1 cytochrome C [Burkholderia ubonensis]
WP_059965520.1 cytochrome C [Burkholderia ubonensis]
WP_059733305.1 cytochrome C [Burkholderia ubonensis]
WP_059849627.1 cytochrome C [Burkholderia ubonensis]
WP_060165437.1 cytochrome C [Burkholderia ubonensis]
WP_059712923.1 cytochrome C [Burkholderia ubonensis]
WP_059737856.1 cytochrome C [Burkholderia ubonensis]
WP_059659999.1 cytochrome C [Burkholderia ubonensis]
WP_059924960.1 cytochrome C [Burkholderia ubonensis]
WP_059777863.1 cytochrome C [Burkholderia ubonensis]
WP_060058770.1 cytochrome C [Burkholderia ubonensis]
WP_059776014.1 cytochrome C [Burkholderia ubonensis]
WP_060123897.1 cytochrome C [Burkholderia ubonensis]
WP_060003887.1 cytochrome C [Burkholderia ubonensis]
WP_071753220.1 cytochrome C [Burkholderia ubonensis]
WP_069271183.1 cytochrome C [Burkholderia ubonensis]
WP_060168536.1 cytochrome C [Burkholderia ubonensis]
WP_059937950.1 cytochrome C [Burkholderia ubonensis]
WP_059651010.1 cytochrome C [Burkholderia ubonensis]
WP_059952547.1 cytochrome C [Burkholderia ubonensis]
WP_059727989.1 cytochrome C [Burkholderia ubonensis]
WP_060328321.1 cytochrome C [Burkholderia ubonensis]
WP_059872364.1 cytochrome C [Burkholderia ubonensis]
WP_059997503.1 cytochrome C [Burkholderia ubonensis]
WP_071773630.1 cytochrome C [Burkholderia ubonensis]
WP_059828379.1 cytochrome C [Burkholderia ubonensis]
WP_060088886.1 cytochrome C [Burkholderia ubonensis]
WP_060248705.1 cytochrome C [Burkholderia ubonensis]
WP_036661765.1 cytochrome C [Pandoraea sp. SD6-2]
WP_059573013.1 cytochrome C [Burkholderia sp. TSV86]
WP_006027348.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_069235777.1 cytochrome C [Burkholderia sp. Bp7605]
WP_059514380.1 cytochrome C [Burkholderia sp. TSV85]
WP_063533919.1 cytochrome C [Burkholderia sp. MSMB1589WGS]
WP_059929957.1 MULTISPECIES: cytochrome C [pseudomallei group]
WP_060819951.1 cytochrome C [Burkholderia sp. BDU19]
WP_059646659.1 MULTISPECIES: cytochrome C [pseudomallei group]
WP_038746875.1 cytochrome C [Burkholderia sp. ABCPW 111]
WP_059669953.1 cytochrome C [Burkholderia sp. MSMB1498]
WP_038801472.1 cytochrome C [Burkholderia oklahomensis]
WP_060356553.1 cytochrome C [Burkholderia sp. MSMB617WGS]
WP_059582413.1 MULTISPECIES: cytochrome C [pseudomallei group]
WP_085508044.1 cytochrome C [Burkholderia pseudomallei]
WP_010118598.1 cytochrome C [Burkholderia oklahomensis]
WP_059597682.1 cytochrome C [Burkholderia sp. BDU6]
WP_025405690.1 cytochrome C [Burkholderia thailandensis]
WP_038742304.1 cytochrome C [Burkholderia pseudomallei]
WP_009900297.1 cytochrome C [Burkholderia thailandensis]（配列番号１１）
WP_004532924.1 cytochrome C [Burkholderia pseudomallei]
WP_058034623.1 cytochrome C [Burkholderia pseudomallei]
WP_038758421.1 cytochrome C [Burkholderia pseudomallei]
WP_038760788.1 cytochrome C [Burkholderia pseudomallei]
WP_004540659.1 cytochrome C [Burkholderia pseudomallei]
WP_085547264.1 cytochrome C [Burkholderia pseudomallei]
WP_076883397.1 cytochrome C [Burkholderia pseudomallei]
WP_059473995.1 cytochrome C [Burkholderia sp. BDU5]
WP_041191039.1 cytochrome C [Burkholderia pseudomallei]
WP_043296725.1 cytochrome C [Burkholderia thailandensis]
WP_063597172.1 cytochrome C [Burkholderia pseudomallei]
WP_066496408.1 cytochrome C [Burkholderia sp. BDU8]
WP_038799895.1 cytochrome C [Burkholderia pseudomallei]
WP_004539114.1 cytochrome C [Burkholderia pseudomallei]
WP_004524074.1 cytochrome C [Burkholderia pseudomallei]
WP_017881863.1 cytochrome C [Burkholderia pseudomallei]
WP_009897184.1 cytochrome C [Burkholderia thailandensis]
WP_004536717.1 cytochrome C [Burkholderia pseudomallei]
WP_076885712.1 cytochrome C [Burkholderia pseudomallei]
WP_066570748.1 cytochrome C [Burkholderia sp. ABCPW 14]
WP_076903220.1 cytochrome C [Burkholderia pseudomallei]
WP_076936464.1 cytochrome C [Burkholderia pseudomallei]
WP_076893617.1 cytochrome C [Burkholderia pseudomallei]
WP_043299328.1 cytochrome C [Burkholderia pseudomallei]
WP_038785043.1 cytochrome C [Burkholderia pseudomallei]
WP_041220968.1 cytochrome C [Burkholderia pseudomallei]
WP_076891198.1 cytochrome C [Burkholderia pseudomallei]
WP_076883909.1 cytochrome C [Burkholderia pseudomallei]
WP_004528232.1 cytochrome C [Burkholderia pseudomallei]
WP_085539130.1 cytochrome C [Burkholderia pseudomallei]
WP_038784065.1 cytochrome C [Burkholderia pseudomallei]
WP_038760183.1 cytochrome C [Burkholderia pseudomallei]
WP_038730591.1 cytochrome C [Burkholderia pseudomallei]
WP_038765827.1 cytochrome C [Burkholderia pseudomallei]
WP_076855330.1 cytochrome C [Burkholderia pseudomallei]
WP_004199672.1 cytochrome C [Burkholderia mallei]
KGC89207.1 cytochrome C family protein [Burkholderia pseudomallei]
EDO93432.1 glucose dehydrogenase, beta subunit [Burkholderia pseudomallei Pasteur 52237]
WP_011205494.1 cytochrome C subunit II [Burkholderia pseudomallei]
KGV82938.1 cytochrome C family protein [Burkholderia pseudomallei MSHR4375]
CDU31054.1 putative cytochrome C subunit II [Burkholderia pseudomallei]
KGV67566.1 cytochrome C family protein [Burkholderia pseudomallei MSHR4299]
EDK84065.1 cytochrome C family protein [Burkholderia mallei 2002721280]
WP_074287454.1 cytochrome C [Burkholderia sp. GAS332]
WP_035557403.1 cytochrome C [Burkholderia sp. 9120]
WP_084534186.1 cytochrome C [Paraburkholderia dilworthii]
WP_017774215.1 cytochrome C [Paraburkholderia kururiensis]
WP_030100524.1 cytochrome C [Burkholderia sp. K24]
WP_028194542.1 cytochrome C [Paraburkholderia fungorum]
WP_046573324.1 cytochrome C [Paraburkholderia fungorum]
WP_042300635.1 cytochrome C [Paraburkholderia kururiensis]
WP_084166897.1 cytochrome C [Paraburkholderia caledonica]
SDI05126.1 cytochrome C, mono-and diheme variants [Paraburkholderia phenazinium]WP_051120977.1 cytochrome C [Paraburkholderia bryophila]
WP_073428494.1 MULTISPECIES: cytochrome C [Burkholderiaceae]
WP_074300113.1 cytochrome C [Paraburkholderia phenazinium]
WP_074768708.1 cytochrome C [Paraburkholderia fungorum]
WP_075465130.1 cytochrome C [Ralstonia solanacearum]
WP_039597687.1 cytochrome C [Ralstonia sp. A12]
WP_055334967.1 cytochrome C [Ralstonia solanacearum]
WP_078223437.1 cytochrome C [blood disease bacterium A2-HR MARDI]
WP_013213209.1 cytochrome C [Ralstonia solanacearum]
WP_063393008.1 cytochrome C [Ralstonia mannitolilytica]
WP_003265144.1 cytochrome C, partial [Ralstonia solanacearum]（配列番号１２）
WP_039568931.1 cytochrome C [Ralstonia solanacearum]
WP_045786290.1 cytochrome C [Ralstonia mannitolilytica]
WP_064802148.1 cytochrome C [Ralstonia insidiosa]
WP_021195198.1 MULTISPECIES: cytochrome C [Ralstonia]
WP_004629446.1 cytochrome C, mono- and diheme variants family [Ralstonia pickettii]
WP_012435095.1 cytochrome C [Ralstonia pickettii]
WP_003279246.1 cytochrome C [Ralstonia solanacearum]
WP_048931829.1 cytochrome C [Ralstonia sp. MD27]
WP_027677928.1 cytochrome C [Ralstonia sp. UNC404CL21Col]
WP_012761509.1 cytochrome C [Ralstonia pickettii]（配列番号１３）
WP_045204557.1 cytochrome C [Burkholderiaceae bacterium 26]
CUV46938.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum]
WP_024973348.1 cytochrome C [Ralstonia pickettii]
AKZ27209.1 cytochrome C [Ralstonia solanacearum]
WP_020749403.1 oxidoreductase dehydrogenase (cytochrome C subunit) [Ralstonia solanacearum]
WP_024976325.1 cytochrome C [Ralstonia pickettii]
WP_049842155.1 cytochrome C [Ralstonia solanacearum]
WP_028852719.1 cytochrome C [Ralstonia solanacearum]
WP_071895582.1 cytochrome C [Ralstonia solanacearum]
SFP41323.1 cytochrome C, mono-and diheme variants [Ralstonia sp. NFACC01]
WP_064477581.1 cytochrome C [Ralstonia solanacearum]
WP_058908222.1 cytochrome C [Ralstonia solanacearum]
WP_009238766.1 MULTISPECIES: cytochrome C [Ralstonia]
CUV21878.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum]
WP_011000726.1 cytochrome C [Ralstonia solanacearum]
WP_071507651.1 cytochrome C [Ralstonia solanacearum]
WP_065857157.1 cytochrome C [Ralstonia pickettii]
WP_019717689.1 cytochrome C [Ralstonia solanacearum]
WP_071012822.1 cytochrome C [Ralstonia solanacearum]
WP_020831436.1 2-Keto-D-gluconate dehydrogenase [Ralstonia solanacearum]
CUV55668.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum] WP_006403391.1 MULTISPECIES: cytochrome C [Burkholderia]
AAQ06608.1 glucose dehydrogenase beta subunit [Burkholderia cepacia]
WP_006396899.1 cytochrome C [Burkholderia multivorans] (SEQ ID NO: 8)
SAJ95286.1 Gluconate 2-dehydrogenase (acceptor) [Burkholderia multivorans]
WP_006412653.1 cytochrome C [Burkholderia multivorans]
WP_060041792.1 cytochrome C [Burkholderia multivorans]
WP_060112921.1 cytochrome C [Burkholderia multivorans]
WP_060182288.1 cytochrome C [Burkholderia multivorans]
WP_060151834.1 cytochrome C [Burkholderia multivorans]
WP_048804658.1 cytochrome C [Burkholderia multivorans]
WP_059786407.1 cytochrome C [Burkholderia multivorans]
WP_035955019.1 cytochrome C [Burkholderia multivorans]
WP_059585013.1 cytochrome C [Burkholderia anthina]
WP_006482958.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex] (SEQ ID NO: 6)
WP_048988065.1 cytochrome C [Burkholderia cenocepacia] (SEQ ID NO: 7)
WP_084751507.1 cytochrome C [Burkholderia cenocepacia]
WP_077186512.1 cytochrome C [Burkholderia cenocepacia]
WP_069351905.1 cytochrome C [Burkholderia cenocepacia]
WP_059783319.1 cytochrome C [Burkholderia sp. NRF60-BP8]
WP_060263394.1 cytochrome C [Burkholderia cenocepacia]
WP_059836247.1 cytochrome C [Burkholderia sp. MSMB1835]
WP_059556637.1 cytochrome C [Burkholderia seminalis]
WP_063551853.1 cytochrome C [Burkholderia territorii]
WP_060310848.1 cytochrome C [Burkholderia anthina]
WP_034187893.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_059789576.1 cytochrome C [Burkholderia sp. MSMB1072]
WP_059500343.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_034204695.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_011882359.1 cytochrome C [Burkholderia vietnamiensis]
WP_060968838.1 cytochrome C [Burkholderia anthina]
WP_077204198.1 cytochrome C [Burkholderia cenocepacia]
WP_050012790.1 cytochrome C [Burkholderia cenocepacia]
WP_011547563.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_014724780.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_059453668.1 cytochrome C [Burkholderia vietnamiensis]
WP_034195126.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_027812584.1 cytochrome C [Burkholderia cenocepacia]
WP_059720348.1 cytochrome C [Burkholderia vietnamiensis]
WP_059548127.1 cytochrome C [Burkholderia vietnamiensis]
WP_044843288.1 cytochrome C [Burkholderia sp. USM B20]
WP_059577619.1 cytochrome C [Burkholderia vietnamiensis]
WP_059459380.1 cytochrome C [Burkholderia vietnamiensis]
WP_059451171.1 cytochrome C [Burkholderia territorii]
WP_027780832.1 cytochrome C [Burkholderia cepacia]
WP_077217239.1 cytochrome C [Burkholderia cenocepacia]
WP_059539736.1 cytochrome C [Burkholderia diffusa]
WP_059507572.1 cytochrome C [Burkholderia territorii]
AOJ19136.1 cytochrome C [Burkholderia cenocepacia]
WP_027806162.1 cytochrome C [Burkholderia cenocepacia]
WP_060107812.1 cytochrome C [Burkholderia territorii]
WP_059822261.1 cytochrome C [Burkholderia sp. MSMB1826]
WP_059464697.1 cytochrome C [Burkholderia diffusa]
WP_059240315.1 cytochrome C [Burkholderia cepacia]
WP_059734410.1 cytochrome C [Burkholderia vietnamiensis]
WP_069227164.1 cytochrome C [Burkholderia diffusa]
WP_060118353.1 cytochrome C [Burkholderia territorii]
WP_069617959.1 cytochrome C [Burkholderia sp. A2]
WP_060347293.1 cytochrome C [Burkholderia territorii]
WP_059702517.1 cytochrome C [Burkholderia vietnamiensis]
WP_060126789.1 cytochrome C [Burkholderia territorii]
WP_069260162.1 cytochrome C [Burkholderia metallica]
WP_059978049.1 cytochrome C [Burkholderia territorii]
WP_059691288.1 cytochrome C [Burkholderia sp. RF4-BP95]
WP_040140035.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_011349243.1 cytochrome C [Burkholderia lata]
WP_059533875.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_059547167.1 cytochrome C [Burkholderia latens]
WP_057924955.1 cytochrome C [Burkholderia ambifaria]
WP_059607236.1 cytochrome C [Burkholderia sp. LA-2-3-30-S1-D2]
WP_039351157.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_046548033.1 cytochrome C [Burkholderia contaminans]
WP_006752013.1 cytochrome C [Burkholderia ambifaria]
WP_014899067.1 cytochrome C [Burkholderia cepacia]
WP_006756339.1 cytochrome C [Burkholderia ambifaria]
WP_059819259.1 cytochrome C [Burkholderia sp. MSMB0856]
WP_011354899.1 cytochrome C [Burkholderia lata]
WP_039320755.1 cytochrome C [Burkholderia sp.
WP_035974222.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_071332428.1 cytochrome C [Burkholderia contaminans]
WP_059684703.1 cytochrome C [Burkholderia sp. FL-7-2-10-S1-D7]
WP_048252126.1 cytochrome C [Burkholderia cepacia]
WP_069251790.1 cytochrome C [Burkholderia lata]
WP_011658978.1 cytochrome C [Burkholderia ambifaria]
WP_012366204.1 cytochrome C [Burkholderia ambifaria]
WP_072438780.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_031400525.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_034180248.1 cytochrome C [Burkholderia pyrrocinia]
WP_043184377.1 cytochrome C [Burkholderia cepacia]
WP_059729924.1 cytochrome C [Burkholderia cepacia]
WP_059713002.1 cytochrome C [Burkholderia ubonensis] (SEQ ID NO: 9)
WP_065501791.1 cytochrome C [Burkholderia stabilis]
WP_059615074.1 cytochrome C [Burkholderia ubonensis]
WP_085037374.1 cytochrome C [Burkholderia sp. CAMPA 1040]
WP_060359188.1 cytochrome C [Burkholderia cepacia]
WP_060333590.1 cytochrome C [Burkholderia ubonensis]
WP_059522548.1 cytochrome C [Burkholderia cepacia]
WP_059766841.1 cytochrome C [Burkholderia ubonensis]
WP_059637760.1 cytochrome C [Burkholderia ubonensis]
WP_040131620.1 MULTISPECIES: cytochrome C [Burkholderia cepacia complex]
WP_060052348.1 cytochrome C [Burkholderia cepacia]
WP_021162032.1 MULTISPECIES: glucose dehydrogenase [Burkholderia]
WP_059666051.1 cytochrome C [Burkholderia cepacia]
WP_060375463.1 cytochrome C [Burkholderia cepacia]
WP_060371861.1 cytochrome C [Burkholderia cepacia]
WP_059697397.1 cytochrome C [Burkholderia cepacia]
WP_047903070.1 cytochrome C [Burkholderia pyrrocinia]
WP_027791850.1 cytochrome C [Burkholderia cepacia]
WP_059552416.1 cytochrome C [Burkholderia ubonensis]
WP_060232513.1 cytochrome C [Burkholderia cepacia]
WP_059687063.1 cytochrome C [Burkholderia cepacia]
WP_060226065.1 cytochrome C [Burkholderia cepacia]
WP_060125713.1 cytochrome C [Burkholderia cepacia]
WP_059923490.1 cytochrome C [Burkholderia stagnalis] (SEQ ID NO: 10)
WP_059565670.1 cytochrome C [Burkholderia stagnalis]
OJD09200.1 cytochrome C [Burkholderia sp. DNA89]
WP_060237338.1 cytochrome C [Burkholderia pseudomultivorans]
WP_059707234.1 cytochrome C [Burkholderia cepacia]
WP_060362538.1 cytochrome C [Burkholderia stagnalis]
WP_060159353.1 cytochrome C [Burkholderia stagnalis]
WP_069748432.1 cytochrome C [Burkholderia stabilis]
WP_059677336.1 cytochrome C [Burkholderia cepacia]
WP_059730424.1 cytochrome C [Burkholderia ubonensis]
WP_059813883.1 cytochrome C [Burkholderia cepacia]
WP_059993161.1 cytochrome C [Burkholderia stagnalis]
WP_060236459.1 cytochrome C [Burkholderia ubonensis]
WP_076476708.1 cytochrome C [Burkholderia ubonensis]
WP_010089725.1 cytochrome C [Burkholderia ubonensis]
WP_060195541.1 cytochrome C [Burkholderia ubonensis]
WP_059971786.1 cytochrome C [Burkholderia pyrrocinia]
WP_060367052.1 cytochrome C [Burkholderia ubonensis]
WP_060016611.1 cytochrome C [Burkholderia ubonensis]
WP_059924260.1 cytochrome C [Burkholderia ubonensis]
WP_060048488.1 cytochrome C [Burkholderia ubonensis]
WP_059892434.1 cytochrome C [Burkholderia ubonensis]
WP_059488090.1 cytochrome C [Burkholderia ubonensis]
WP_059483872.1 cytochrome C [Burkholderia ubonensis]
WP_059865488.1 cytochrome C [Burkholderia ubonensis]
WP_059633519.1 cytochrome C [Burkholderia ubonensis]
WP_060287380.1 cytochrome C [Burkholderia ubonensis]
WP_059853060.1 cytochrome C [Burkholderia ubonensis]
WP_059968663.1 cytochrome C [Burkholderia ubonensis]
WP_059752176.1 cytochrome C [Burkholderia ubonensis]
WP_059532869.1 cytochrome C [Burkholderia ubonensis]
WP_059660535.1 cytochrome C [Burkholderia ubonensis]
WP_059590294.1 cytochrome C [Burkholderia ubonensis]
WP_026043943.1 cytochrome C [Burkholderia pyrrocinia]
WP_060264220.1 cytochrome C [Burkholderia ubonensis]
WP_060019510.1 cytochrome C [Burkholderia ubonensis]
WP_059946330.1 cytochrome C [Burkholderia ubonensis]
WP_042588017.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_084908871.1 cytochrome C [[Pseudomonas] mesoacidophila]
WP_071751088.1 cytochrome C [Burkholderia ubonensis]
WP_060094261.1 cytochrome C [Burkholderia ubonensis]
WP_059885808.1 cytochrome C [Burkholderia ubonensis]
WP_059878406.1 cytochrome C [Burkholderia ubonensis]
WP_071852856.1 cytochrome C [Burkholderia ubonensis]
WP_060161154.1 cytochrome C [Burkholderia ubonensis]
WP_059796027.1 cytochrome C [Burkholderia ubonensis]
WP_059835010.1 cytochrome C [Burkholderia ubonensis]
WP_059911385.1 cytochrome C [Burkholderia ubonensis]
WP_059744627.1 cytochrome C [Burkholderia ubonensis]
WP_045565069.1 cytochrome C [Burkholderia ubonensis]
WP_060276465.1 cytochrome C [Burkholderia ubonensis]
WP_060052180.1 cytochrome C [Burkholderia ubonensis]
WP_059877204.1 cytochrome C [Burkholderia ubonensis]
WP_059918860.1 cytochrome C [Burkholderia ubonensis]
WP_059674217.1 cytochrome C [Burkholderia ubonensis]
WP_059626644.1 cytochrome C [Burkholderia ubonensis]
WP_059706954.1 cytochrome C [Burkholderia ubonensis]
WP_059610359.1 cytochrome C [Burkholderia ubonensis]
WP_071751781.1 cytochrome C [Burkholderia ubonensis]
WP_060345076.1 cytochrome C [Burkholderia ubonensis]
WP_060056668.1 cytochrome C [Burkholderia ubonensis]
WP_059867004.1 cytochrome C [Burkholderia ubonensis]
WP_059846198.1 cytochrome C [Burkholderia ubonensis]
WP_059956839.1 cytochrome C [Burkholderia ubonensis]
WP_059801215.1 cytochrome C [Burkholderia ubonensis]
WP_060374144.1 cytochrome C [Burkholderia ubonensis]
WP_060228155.1 cytochrome C [Burkholderia ubonensis]
WP_060449283.1 cytochrome C [Burkholderia ubonensis]
WP_060088315.1 cytochrome C [Burkholderia ubonensis]
WP_060141594.1 cytochrome C [Burkholderia ubonensis]
WP_059965520.1 cytochrome C [Burkholderia ubonensis]
WP_059733305.1 cytochrome C [Burkholderia ubonensis]
WP_059849627.1 cytochrome C [Burkholderia ubonensis]
WP_060165437.1 cytochrome C [Burkholderia ubonensis]
WP_059712923.1 cytochrome C [Burkholderia ubonensis]
WP_059737856.1 cytochrome C [Burkholderia ubonensis]
WP_059659999.1 cytochrome C [Burkholderia ubonensis]
WP_059924960.1 cytochrome C [Burkholderia ubonensis]
WP_059777863.1 cytochrome C [Burkholderia ubonensis]
WP_060058770.1 cytochrome C [Burkholderia ubonensis]
WP_059776014.1 cytochrome C [Burkholderia ubonensis]
WP_060123897.1 cytochrome C [Burkholderia ubonensis]
WP_060003887.1 cytochrome C [Burkholderia ubonensis]
WP_071753220.1 cytochrome C [Burkholderia ubonensis]
WP_069271183.1 cytochrome C [Burkholderia ubonensis]
WP_060168536.1 cytochrome C [Burkholderia ubonensis]
WP_059937950.1 cytochrome C [Burkholderia ubonensis]
WP_059651010.1 cytochrome C [Burkholderia ubonensis]
WP_059952547.1 cytochrome C [Burkholderia ubonensis]
WP_059727989.1 cytochrome C [Burkholderia ubonensis]
WP_060328321.1 cytochrome C [Burkholderia ubonensis]
WP_059872364.1 cytochrome C [Burkholderia ubonensis]
WP_059997503.1 cytochrome C [Burkholderia ubonensis]
WP_071773630.1 cytochrome C [Burkholderia ubonensis]
WP_059828379.1 cytochrome C [Burkholderia ubonensis]
WP_060088886.1 cytochrome C [Burkholderia ubonensis]
WP_060248705.1 cytochrome C [Burkholderia ubonensis]
WP_036661765.1 cytochrome C [Pandoraea sp. SD6-2]
WP_059573013.1 cytochrome C [Burkholderia sp. TSV86]
WP_006027348.1 MULTISPECIES: cytochrome C [Burkholderia]
WP_069235777.1 cytochrome C [Burkholderia sp. Bp7605]
WP_059514380.1 cytochrome C [Burkholderia sp. TSV85]
WP_063533919.1 cytochrome C [Burkholderia sp. MSMB1589WGS]
WP_059929957.1 MULTISPECIES: cytochrome C [pseudomallei group]
WP_060819951.1 cytochrome C [Burkholderia sp. BDU19]
WP_059646659.1 MULTISPECIES: cytochrome C [pseudomallei group]
WP_038746875.1 cytochrome C [Burkholderia sp. ABCPW 111]
WP_059669953.1 cytochrome C [Burkholderia sp. MSMB1498]
WP_038801472.1 cytochrome C [Burkholderia oklahomensis]
WP_060356553.1 cytochrome C [Burkholderia sp. MSMB617WGS]
WP_059582413.1 MULTISPECIES: cytochrome C [pseudomallei group]
WP_085508044.1 cytochrome C [Burkholderia pseudomallei]
WP_010118598.1 cytochrome C [Burkholderia oklahomensis]
WP_059597682.1 cytochrome C [Burkholderia sp. BDU6]
WP_025405690.1 cytochrome C [Burkholderia thailandensis]
WP_038742304.1 cytochrome C [Burkholderia pseudomallei]
WP_009900297.1 cytochrome C [Burkholderia thailandensis] (SEQ ID NO: 11)
WP_004532924.1 cytochrome C [Burkholderia pseudomallei]
WP_058034623.1 cytochrome C [Burkholderia pseudomallei]
WP_038758421.1 cytochrome C [Burkholderia pseudomallei]
WP_038760788.1 cytochrome C [Burkholderia pseudomallei]
WP_004540659.1 cytochrome C [Burkholderia pseudomallei]
WP_085547264.1 cytochrome C [Burkholderia pseudomallei]
WP_076883397.1 cytochrome C [Burkholderia pseudomallei]
WP_059473995.1 cytochrome C [Burkholderia sp. BDU5]
WP_041191039.1 cytochrome C [Burkholderia pseudomallei]
WP_043296725.1 cytochrome C [Burkholderia thailandensis]
WP_063597172.1 cytochrome C [Burkholderia pseudomallei]
WP_066496408.1 cytochrome C [Burkholderia sp. BDU8]
WP_038799895.1 cytochrome C [Burkholderia pseudomallei]
WP_004539114.1 cytochrome C [Burkholderia pseudomallei]
WP_004524074.1 cytochrome C [Burkholderia pseudomallei]
WP_017881863.1 cytochrome C [Burkholderia pseudomallei]
WP_009897184.1 cytochrome C [Burkholderia thailandensis]
WP_004536717.1 cytochrome C [Burkholderia pseudomallei]
WP_076885712.1 cytochrome C [Burkholderia pseudomallei]
WP_066570748.1 cytochrome C [Burkholderia sp. ABCPW 14]
WP_076903220.1 cytochrome C [Burkholderia pseudomallei]
WP_076936464.1 cytochrome C [Burkholderia pseudomallei]
WP_076893617.1 cytochrome C [Burkholderia pseudomallei]
WP_043299328.1 cytochrome C [Burkholderia pseudomallei]
WP_038785043.1 cytochrome C [Burkholderia pseudomallei]
WP_041220968.1 cytochrome C [Burkholderia pseudomallei]
WP_076891198.1 cytochrome C [Burkholderia pseudomallei]
WP_076883909.1 cytochrome C [Burkholderia pseudomallei]
WP_004528232.1 cytochrome C [Burkholderia pseudomallei]
WP_085539130.1 cytochrome C [Burkholderia pseudomallei]
WP_038784065.1 cytochrome C [Burkholderia pseudomallei]
WP_038760183.1 cytochrome C [Burkholderia pseudomallei]
WP_038730591.1 cytochrome C [Burkholderia pseudomallei]
WP_038765827.1 cytochrome C [Burkholderia pseudomallei]
WP_076855330.1 cytochrome C [Burkholderia pseudomallei]
WP_004199672.1 cytochrome C [Burkholderia mallei]
KGC89207.1 cytochrome C family protein [Burkholderia pseudomallei]
EDO93432.1 glucose dehydrogenase, beta subunit [Burkholderia pseudomallei Pasteur 52237]
WP_011205494.1 cytochrome C subunit II [Burkholderia pseudomallei]
KGV82938.1 cytochrome C family protein [Burkholderia pseudomallei MSHR4375]
CDU31054.1 putative cytochrome C subunit II [Burkholderia pseudomallei]
KGV67566.1 cytochrome C family protein [Burkholderia pseudomallei MSHR4299]
EDK84065.1 cytochrome C family protein [Burkholderia mallei 2002721280]
WP_074287454.1 cytochrome C [Burkholderia sp. GAS332]
WP_035557403.1 cytochrome C [Burkholderia sp. 9120]
WP_084534186.1 cytochrome C [Paraburkholderia dilworthii]
WP_017774215.1 cytochrome C [Paraburkholderia kururiensis]
WP_030100524.1 cytochrome C [Burkholderia sp. K24]
WP_028194542.1 cytochrome C [Paraburkholderia fungorum]
WP_046573324.1 cytochrome C [Paraburkholderia fungorum]
WP_042300635.1 cytochrome C [Paraburkholderia kururiensis]
WP_084166897.1 cytochrome C [Paraburkholderia caledonica]
SDI05126.1 cytochrome C, mono-and diheme variants [Paraburkholderia phenazinium]WP_051120977.1 cytochrome C [Paraburkholderia bryophila]
WP_073428494.1 MULTISPECIES: cytochrome C [Burkholderiaceae]
WP_074300113.1 cytochrome C [Paraburkholderia phenazinium]
WP_074768708.1 cytochrome C [Paraburkholderia fungorum]
WP_075465130.1 cytochrome C [Ralstonia solanacearum]
WP_039597687.1 cytochrome C [Ralstonia sp.
WP_055334967.1 cytochrome C [Ralstonia solanacearum]
WP_078223437.1 cytochrome C [blood disease bacterium A2-HR MARDI]
WP_013213209.1 cytochrome C [Ralstonia solanacearum]
WP_063393008.1 cytochrome C [Ralstonia mannitolilytica]
WP_003265144.1 cytochrome C, partial [Ralstonia solanacearum] (SEQ ID NO: 12)
WP_039568931.1 cytochrome C [Ralstonia solanacearum]
WP_045786290.1 cytochrome C [Ralstonia mannitolilytica]
WP_064802148.1 cytochrome C [Ralstonia insidiosa]
WP_021195198.1 MULTISPECIES: cytochrome C [Ralstonia]
WP_004629446.1 cytochrome C, mono- and diheme variants family [Ralstonia pickettii]
WP_012435095.1 cytochrome C [Ralstonia pickettii]
WP_003279246.1 cytochrome C [Ralstonia solanacearum]
WP_048931829.1 cytochrome C [Ralstonia sp. MD27]
WP_027677928.1 cytochrome C [Ralstonia sp. UNC404CL21Col]
WP_012761509.1 cytochrome C [Ralstonia pickettii] (SEQ ID NO: 13)
WP_045204557.1 cytochrome C [Burkholderiaceae bacterium 26]
CUV46938.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum]
WP_024973348.1 cytochrome C [Ralstonia pickettii]
AKZ27209.1 cytochrome C [Ralstonia solanacearum]
WP_020749403.1 oxidoreductase dehydrogenase (cytochrome C subunit) [Ralstonia solanacearum]
WP_024976325.1 cytochrome C [Ralstonia pickettii]
WP_049842155.1 cytochrome C [Ralstonia solanacearum]
WP_028852719.1 cytochrome C [Ralstonia solanacearum]
WP_071895582.1 cytochrome C [Ralstonia solanacearum]
SFP41323.1 cytochrome C, mono-and diheme variants [Ralstonia sp.
WP_064477581.1 cytochrome C [Ralstonia solanacearum]
WP_058908222.1 cytochrome C [Ralstonia solanacearum]
WP_009238766.1 MULTISPECIES: cytochrome C [Ralstonia]
CUV21878.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum]
WP_011000726.1 cytochrome C [Ralstonia solanacearum]
WP_071507651.1 cytochrome C [Ralstonia solanacearum]
WP_065857157.1 cytochrome C [Ralstonia pickettii]
WP_019717689.1 cytochrome C [Ralstonia solanacearum]
WP_071012822.1 cytochrome C [Ralstonia solanacearum]
WP_020831436.1 2-Keto-D-gluconate dehydrogenase [Ralstonia solanacearum]
CUV55668.1 Gluconate 2-dehydrogenase cytochrome C subunit [Ralstonia solanacearum]

本発明の変異型シトクロムＣタンパク質は、上述したような配列番号４と６０％以上の配列同一性を有するアミノ酸配列、例えば、配列番号６～１３のいずれかのアミノ酸配列において、アミノ末端側から数えて１、２番目の第１および第２のヘム結合ドメインが欠損するように改変されたものも含まれる。例えば、配列番号６～１３のいずれかのアミノ酸配列において、第１および第２のヘム結合ドメインが欠損したもの、配列番号６～１３のいずれかのアミノ酸配列において、第１、第２のヘム結合ドメインを含む領域（配列番号４のアミノ酸番号４３～１９５に相当する領域）が欠損したものでもよい。 The mutant cytochrome C protein of the present invention has an amino acid sequence having 60% or more sequence identity with SEQ ID NO: 4 as described above, for example, the amino acid sequence of any one of SEQ ID NOS: 6 to 13, counted from the amino terminal side. Also included are those modified to lack the first and second heme-binding domains. For example, the amino acid sequence of any of SEQ ID NOS: 6 to 13 lacking the first and second heme-binding domains, the amino acid sequence of any of SEQ ID NOS: 6 to 13, the first and second heme-binding A region containing a domain (region corresponding to amino acid numbers 43 to 195 of SEQ ID NO:4) may be deleted.

なお、改変のもととなる配列番号６～１３のアミノ酸配列は改変部位および第３のヘム結合ドメイン以外において、１または数個のアミノ酸が置換、欠失、付加または挿入された配列でもよい。 The amino acid sequences of SEQ ID NOs: 6 to 13 to be modified may be sequences in which one or several amino acids are substituted, deleted, added or inserted outside the modification site and the third heme-binding domain.

したがって、本発明の変異型シトクロムＣタンパク質は、電子授受機能を保持する限り、配列番号６～１３のいずれかのアミノ酸配列において第１および第２のヘム結合ドメインが欠損したアミノ酸配列、または、配列番号６～１３のいずれかのアミノ酸配列において第１および第２のヘム結合ドメインを含む領域（配列番号４のアミノ酸番号４３～１９５に相当する領域）が欠損したアミノ酸配列において、さらに、第３のヘム結合ドメイン以外の位置で、１または数個のアミノ酸が置換、欠失、付加または挿入された配列を有するタンパク質でもよい。ここで、「１又は数個」とは、例えば、１～２０個、好ましくは１～１０個、より好ましくは１～８個、さらに好ましくは１～５個、特に好ましくは１～３個である。また、置換は保存的な置換が好ましく、「保存的置換」とは、酸性アミノ酸同士の置換、中性アミノ酸同士の置換、塩基性アミノ酸同士の置換など、性質が類似したアミノ酸同士の置換をいう。 Therefore, the mutant cytochrome C protein of the present invention is an amino acid sequence lacking the first and second heme-binding domains in any of the amino acid sequences of SEQ ID NOs: 6 to 13, or the sequence In the amino acid sequence lacking the regions containing the first and second heme-binding domains (regions corresponding to amino acid numbers 43 to 195 of SEQ ID NO: 4) in any of the amino acid sequences of numbers 6 to 13, the third A protein having a sequence in which one or several amino acids are substituted, deleted, added or inserted at a position other than the heme-binding domain may also be used. Here, "one or several" means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 8, still more preferably 1 to 5, particularly preferably 1 to 3. be. In addition, substitutions are preferably conservative substitutions, and "conservative substitutions" refer to substitutions between amino acids with similar properties, such as substitutions between acidic amino acids, substitutions between neutral amino acids, and substitutions between basic amino acids. .

＜酸化還元酵素・シトクロム複合体＞
本発明の変異型シトクロムタンパク質は、酸化還元酵素の触媒サブユニットとともに使用することができ、酸化還元反応により生じた電子を受け取り、その電子を電極へ渡すことができる。したがって、本発明の変異型シトクロムタンパク質を含む酸化還元酵素・シトクロム複合体は電気化学センサとして使用できる。電気化学センサは好ましくは変異型シトクロムタンパク質とグルコース酸化還元酵素を含むグルコースセンサである。 <Oxidoreductase/cytochrome complex>
Mutant cytochrome proteins of the invention can be used with catalytic subunits of redox enzymes to accept electrons generated by a redox reaction and transfer the electrons to an electrode. Therefore, the oxidoreductase-cytochrome complex containing the mutant cytochrome protein of the present invention can be used as an electrochemical sensor. The electrochemical sensor is preferably a glucose sensor comprising a mutant cytochrome protein and glucose oxidoreductase.

＜酸化還元酵素＞
酸化還元酵素は、測定対象物質を酸化還元しうる酵素であればよいが、触媒サブユニットとして、ピロロキノリンキノン（PQQ）、フラビンアデニンジヌクレオチド（FAD）のうち少なくとも一方を含む酵素の触媒サブユニットを挙げることができる。例えば、PQQを含
む酸化還元酵素として、PQQグルコースデヒドロゲナーゼ（PQQGDH）が挙げられ、FADを含む酸化還元酵素として、FADを含んだαサブユニットを持つシトクロムグルコースデヒド
ロゲナーゼ（CyGDH）、グルコースオキシダーゼ（GOD）が挙げられる。その他、コレステロールオキシダーゼ、キノヘムエタノールデヒドロゲナーゼ（QHEDH (PQQ Ethanol dh)、ソルビトールデヒドロゲナーゼ（Sorbitol DH）、Ｄ-フルクトースデヒドロゲナーゼ（Fructose DH）、セロビオースデヒドロゲナーゼ、乳酸デヒドロゲナーゼが挙げられる。 <Oxidoreductase>
The oxidoreductase may be an enzyme capable of oxidizing or reducing a substance to be measured, and the catalytic subunit of an enzyme containing at least one of pyrroloquinoline quinone (PQQ) and flavin adenine dinucleotide (FAD) as a catalytic subunit. can be mentioned. For example, oxidoreductases containing PQQ include PQQ glucose dehydrogenase (PQQGDH), and oxidoreductases containing FAD include cytochrome glucose dehydrogenase (CyGDH) with an α subunit containing FAD and glucose oxidase (GOD). mentioned. Other examples include cholesterol oxidase, quinohem ethanol dehydrogenase (QHEDH (PQQ Ethanol dh), sorbitol dehydrogenase (Sorbitol DH), D-fructose dehydrogenase (Fructose DH), cellobiose dehydrogenase, and lactate dehydrogenase.

この中ではグルコースデヒドロゲナーゼ（ＧＤＨ）が好ましい。
グルコースデヒドロゲナーゼはグルコースデヒドロゲナーゼ活性を有する限り特に制限されず、公知のものを含め種々の生物に由来するグルコースデヒドロゲナーゼ触媒サブユニットが使用できる。変異型シトクロムタンパク質と同じ微生物由来とすることもできるが、具体的には、例えば、ブルクホリデリア・セパシアＫＳ１株由来の配列番号３のアミノ酸配列を有するαサブユニットタンパク質が使用できる。しかし、ＧＤＨのαサブユニットとして機能し得る限り、配列番号３のアミノ酸配列において、１又は数個のアミノ酸残基が置換、欠失、挿入、又は付加されたアミノ酸配列を有するタンパク質であってもよい。また、ＧＤＨのαサブユニットとして機能し得る限り、ＫＳ１株以外のαサブユニットのアミノ酸配列において、１又は数個のアミノ酸残基が置換、欠失、挿入、又は付加されたアミノ酸配列を有するタンパク質であってもよい。前記「１又は数個」とは、好ましくは１～２０個、より好ましくは１～１０個、特に好ましくは１～５個である。なお、グルコースデヒドロゲナーゼのαサブユニットタンパク質の酵素活性や基質特異性を向上させる変異は数多く知られており、それらの変異を有するグルコースデヒドロゲナーゼのαサブユニットタンパク質が使用できる。 Among these, glucose dehydrogenase (GDH) is preferred.
Glucose dehydrogenase is not particularly limited as long as it has glucose dehydrogenase activity, and glucose dehydrogenase catalytic subunits derived from various organisms including known ones can be used. Although it can be derived from the same microorganism as the mutant cytochrome protein, specifically, for example, an α subunit protein having the amino acid sequence of SEQ ID NO: 3 derived from Burchholideria cepacia KS1 strain can be used. However, as long as it can function as the α subunit of GDH, even if it is a protein having an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 3 good. Also, a protein having an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence of an α subunit other than the KS1 strain, as long as it can function as an α subunit of GDH. may be The "one or several" is preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5. Many mutations that improve the enzymatic activity and substrate specificity of the α-subunit protein of glucose dehydrogenase are known, and the α-subunit protein of glucose dehydrogenase having these mutations can be used.

また、グルコースデヒドロゲナーゼ複合体は、本発明の変異型シトクロムタンパク質と触媒サブユニットに加え、γサブユニットをさらに含んでもよい。γサブユニットとしてはγサブユニットとして機能する限り特に制限されず、公知のものを含め種々の生物に由来するγサブユニットが使用できる。変異型シトクロムタンパク質と同じ微生物由来とすることもできるが、具体的には、例えば、ブルクホリデリア・セパシアＫＳ１株由来の配列番号２のアミノ酸配列を有するタンパク質が使用できる。しかし、γサブユニットとして機能し得る限り、配列番号２からなるアミノ酸配列において、１又は数個のアミノ酸残基が置換、欠失、挿入、又は付加されたアミノ酸配列を有するタンパク質であってもよい。また、γサブユニットとして機能し得る限り、ＫＳ１株以外のγサブユニットのアミノ酸配列において、１又は数個のアミノ酸残基が置換、欠失、挿入、又は付加されたアミノ酸配列を有するタンパク質であってもよい。前記「１又は数個」とは、好ましくは１～１５個、より好ましくは１～１０個、特に好ましくは１～５個である。尚、γサブユニットとして機能するとは、αサブユニットとともに複合体を形成したときに同複合体のＧＤＨ活性を高める機能をいう。 Also, the glucose dehydrogenase complex may further comprise a γ subunit in addition to the mutant cytochrome protein of the present invention and the catalytic subunit. The γ subunit is not particularly limited as long as it functions as a γ subunit, and γ subunits derived from various organisms including known ones can be used. Although it can be derived from the same microorganism as the mutant cytochrome protein, specifically, for example, a protein having the amino acid sequence of SEQ ID NO: 2 derived from Burchholideria cepacia KS1 strain can be used. However, as long as it can function as a γ subunit, it may be a protein having an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, or added to the amino acid sequence consisting of SEQ ID NO:2. . Also, as long as it can function as a γ subunit, it is a protein having an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, or added to the amino acid sequence of a γ subunit other than the KS1 strain. may The "one or several" is preferably 1 to 15, more preferably 1 to 10, and particularly preferably 1 to 5. In addition, functioning as the γ subunit refers to the function of enhancing the GDH activity of the complex when it forms a complex with the α subunit.

＜ＤＮＡ＞
本発明はまた、変異型シトクロムタンパク質をコードするＤＮＡを提供する。変異型シトクロムタンパク質を含むＤＮＡは変異型シトクロムタンパク質のアミノ酸配列に基づき、その塩基配列が特定されうるが、例えば、野生型シトクロムタンパク質をコードするＤＮＡにおいて、第１および第２ヘム結合ドメインまたはこれらを含む領域が欠損するように塩基配列を改変することで得ることができる。
例えば、ブルクホリデリア・セパシアのシトクロムタンパク質をコードするＤＮＡの具体例として、グルコースデヒドロゲナーゼβサブユニットをコードする、配列番号１の塩基番号２３８６～３６６０からなる塩基配列を含むＤＮＡが挙げられる。また、βサブユニ
ットをコードするＤＮＡは、配列番号１の塩基番号２３８６～３６６０からなる塩基配列を有するＤＮＡに限られず、当該塩基配列の相補配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし、かつ、βサブユニットとして機能し得るタンパク質をコードするＤＮＡであってもよい。
変異型シトクロムタンパク質をコードするＤＮＡとして、例えば、配列番号５の塩基番号３３７２～３６６０もしくは３３２５～３６６０からなる塩基配列を有するＤＮＡや当該塩基配列の相補配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし、かつ、変異型シトクロムタンパク質として機能し得るタンパク質をコードするＤＮＡが挙げられる。 <DNA>
The present invention also provides DNAs encoding mutant cytochrome proteins. The base sequence of the DNA containing the mutant cytochrome protein can be specified based on the amino acid sequence of the mutant cytochrome protein. For example, in the DNA encoding the wild-type cytochrome protein, the first and second heme binding domains or It can be obtained by modifying the base sequence so that the containing region is deleted.
For example, a specific example of the DNA encoding the cytochrome protein of Burchholideria cepacia is a DNA comprising a nucleotide sequence consisting of nucleotide numbers 2386 to 3660 of SEQ ID NO: 1, which encodes glucose dehydrogenase β-subunit. In addition, the DNA encoding the β subunit is not limited to DNA having a nucleotide sequence consisting of base numbers 2386 to 3660 of SEQ ID NO: 1, and hybridizes under stringent conditions with DNA having a sequence complementary to the nucleotide sequence. and a DNA encoding a protein capable of functioning as a β subunit.
As the DNA encoding the mutant cytochrome protein, for example, a DNA having a nucleotide sequence consisting of nucleotide numbers 3372 to 3660 or 3325 to 3660 of SEQ ID NO: 5, or a DNA having a complementary sequence to the nucleotide sequence, and hybridized under stringent conditions. DNA that encodes a protein that can soybean and function as a mutant cytochrome protein is included.

所望の変異を有する変異型シトクロムタンパク質は、シトクロムタンパク質をコードするＤＮＡに、部位特異的変異法によって所望のアミノ酸欠失を導入したり、ＰＣＲ法で特定領域のみを増幅させたりすることによって変異ＤＮＡを構築し、これを適当な発現系を用いて発現させることによって、取得することができる。 A mutant cytochrome protein having a desired mutation can be obtained by introducing a desired amino acid deletion into the DNA encoding the cytochrome protein by site-directed mutagenesis or by amplifying only a specific region by PCR. can be obtained by constructing and expressing it using an appropriate expression system.

変異型シトクロムタンパク質を含む酸化還元酵素・シトクロム複合体を使用する場合、酸化還元酵素の触媒サブユニットをコードするＤＮＡを変異型シトクロムタンパク質をコードするＤＮＡとともに用いることが好ましい。
酸化還元酵素の触媒サブユニットをコードするＤＮＡは特に制限されず、目的に応じて適宜選択して使用することができるが、例えば、グルコースデヒドロゲナーゼαサブユニットをコードするＤＮＡとして、配列番号１の塩基番号７６４～２３８０からなる塩基配列を含むＤＮＡが挙げられる。また、αサブユニット遺伝子は、配列番号１の塩基配列の塩基番号７６４～２３８０からなる塩基配列の相補配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし、かつ、ＧＤＨ活性を有するタンパク質をコードするＤＮＡであってもよい。 When using an oxidoreductase-cytochrome complex containing a mutant cytochrome protein, it is preferable to use DNA encoding the catalytic subunit of the oxidoreductase together with DNA encoding the mutant cytochrome protein.
The DNA encoding the catalytic subunit of the oxidoreductase is not particularly limited and can be appropriately selected and used according to the purpose. A DNA containing a base sequence consisting of numbers 764-2380 is mentioned. In addition, the α subunit gene encodes a protein that hybridizes under stringent conditions with DNA having a sequence complementary to the nucleotide sequence consisting of nucleotide numbers 764 to 2380 of the nucleotide sequence of SEQ ID NO: 1 and has GDH activity. It may be DNA that

酸化還元酵素・シトクロム複合体にグルコースデヒドロゲナーゼγサブユニットを含める場合、酸化還元酵素のγサブユニットをコードするＤＮＡを変異型シトクロムタンパク質をコードするＤＮＡおよび触媒サブユニットをコードするＤＮＡとともに用いることが好ましい。
γサブユニットをコードするＤＮＡの具体例としては、配列番号１の塩基番号２５８～７６１からなる塩基配列を含むＤＮＡが挙げられる。またγサブユニットをコードするＤＮＡは、配列番号１の塩基番号２５８～７６１からなる塩基配列の相補配列を有するＤＮＡとストリンジェントな条件下でハイブリダイズし、かつ、γサブユニットとして機能し得るタンパク質をコードするＤＮＡであってもよい。 When the oxidoreductase-cytochrome complex contains the glucose dehydrogenase γ subunit, it is preferable to use the DNA encoding the γ subunit of the oxidoreductase together with the DNA encoding the mutant cytochrome protein and the DNA encoding the catalytic subunit. .
A specific example of the DNA encoding the γ subunit is a DNA containing a base sequence consisting of nucleotide numbers 258-761 of SEQ ID NO:1. Also, the DNA encoding the γ subunit is a protein that can hybridize under stringent conditions with a DNA having a sequence complementary to the nucleotide sequence consisting of nucleotide numbers 258 to 761 of SEQ ID NO: 1 and that can function as a γ subunit. may be a DNA encoding

前記ストリンジェントな条件としては、好ましくは８０％、より好ましくは９０％以上、特に好ましくは９５％以上の同一性を有するＤＮＡ同士がハイブリダイズする条件、具体的には、ハイブリダイゼーション反応の後、０．１×ＳＳＣ、０．１％ＳＤＳ、６０℃の条件下で洗浄する条件が挙げられる。 The stringent conditions are conditions under which DNAs having an identity of preferably 80%, more preferably 90% or more, and particularly preferably 95% or more are hybridized. Specifically, after the hybridization reaction, Examples of washing conditions include 0.1×SSC, 0.1% SDS, and 60°C.

シトクロムタンパク質をコードするＤＮＡ、酸化還元酵素の触媒サブユニットをコードするＤＮＡ等は、ブルクホルデリア・セパシア等の微生物の染色体ＤＮＡを鋳型とするＰＣＲやハイブリダイゼーション法などによって取得することができる。
ブルクホリデリア・セパシアＫＳ１株のＧＤＨ γサブユニット遺伝子、αサブユニット
遺伝子、及びβサブユニット遺伝子を含む染色体ＤＮＡ断片の塩基配列を配列番号１に示す。この塩基配列には３つのオープンリーディングフレーム（ＯＲＦ）が存在し、５’末端側から、１番目のＯＲＦはγサブユニット（配列番号２）をコードし、２番目のＯＲＦはαサブユニット（配列番号３）をコードし、３番目のＯＲＦはβサブユニット（配列番号４）をコードしている。 DNAs encoding cytochrome proteins, DNAs encoding catalytic subunits of oxidoreductases, and the like can be obtained by PCR, hybridization, or the like using the chromosomal DNA of a microorganism such as Burkholderia cepacia as a template.
SEQ ID NO: 1 shows the nucleotide sequence of the chromosomal DNA fragment containing the GDH γ subunit gene, α subunit gene, and β subunit gene of Burchholideria cepacia strain KS1. There are three open reading frames (ORFs) in this nucleotide sequence. From the 5′ end side, the first ORF encodes the γ subunit (SEQ ID NO: 2) and the second ORF encodes the α subunit (sequence number 3) and the third ORF encodes the β subunit (SEQ ID NO: 4).

変異型シトクロムタンパク質、活性サブユニットはそれぞれ別々にＤＮＡを発現させてもよいし、変異型シトクロムタンパク質をコードするＤＮＡと活性サブユニットをコードするＤＮＡをポリシストロニックに含むＤＮＡを用いて発現させてもよい。 The mutant cytochrome protein and the active subunit may be expressed as separate DNAs, or may be expressed using polycistronic DNA containing the DNA encoding the mutant cytochrome protein and the DNA encoding the active subunit. good too.

＜ベクター＞
変異型シトクロムタンパク質、活性サブユニットの遺伝子の取得、変異の導入、遺伝子の発現等に用いるベクターとしては、宿主微生物で機能するベクターであれば特に制限されないが、例えばエシェリヒア属細菌で機能するベクター、具体的にはｐＴｒｃ９９Ａ、ｐＢＲ３２２、ｐＵＣ１８、ｐＵＣ１１８、ｐＵＣ１９、ｐＵＣ１１９、ｐＡＣＹＣ１８４、ｐＢＢＲ１２２等が挙げられる。遺伝子の発現に用いるプロモーターも宿主に応じて適宜選択できるが、例えばｌａｃ、ｔｒｐ、ｔａｃ、ｔｒｃ、ＰL、ｔｅｔ、ＰｈｏＡ等が
挙げられる。 <Vector>
The vector used for obtaining the gene of the mutant cytochrome protein or active subunit, introducing mutation, expressing the gene, etc. is not particularly limited as long as it is a vector that functions in the host microorganism. Specific examples include pTrc99A, pBR322, pUC18, pUC118, pUC19, pUC119, pACYC184, pBBR122 and the like. Promoters used for gene expression can also be appropriately selected according to the host, and examples thereof include lac, trp, tac, trc, PL, tet, and PhoA.

＜形質転換体＞
上記のようなＤＮＡまたはそれを含むベクターを宿主微生物に導入して形質転換体を得ることで、変異型シトクロムタンパク質や酸化還元酵素・シトクロム複合体を発現させることができる。形質転換は公知の方法を採用でき、例えばカルシウム処理によるコンピテントセル法、プロトプラスト法又はエレクトロポレーション法等が挙げられる。 <Transformant>
A mutant cytochrome protein or an oxidoreductase/cytochrome complex can be expressed by introducing a DNA or a vector containing the DNA as described above into a host microorganism to obtain a transformant. A known method can be employed for transformation, and examples thereof include a competent cell method by calcium treatment, a protoplast method, an electroporation method, and the like.

宿主微生物としてはタンパク質発現に使用されるものであれば特に制限されないが、エシェリヒア・コリ等のエシェリヒア属細菌、バチルス・サブチリス等のバチルス属細菌、サッカロマイセス・セレビシエ、ピキア・パストリス等の酵母、アスペルギルス・ニガー、アスペルギルス・オリゼ等の糸状菌が挙げられるが、これらに限られない。 The host microorganism is not particularly limited as long as it is used for protein expression, and includes bacteria belonging to the genus Escherichia such as Escherichia coli, bacteria belonging to the genus Bacillus such as Bacillus subtilis, yeasts such as Saccharomyces cerevisiae and Pichia pastoris, and Aspergillus. Examples include, but are not limited to, filamentous fungi such as Niger and Aspergillus oryzae.

＜バイオセンサ＞
本発明の変異型シトクロムタンパク質は酸化還元反応を利用する電気化学的バイオセンサに使用できる。バイオセンサの種類は組み合わせる酸化還元酵素の種類によって様々な用途とすることができ、例えば、酸化還元酵素としてグルコース酸化還元酵素を用いる場合はグルコースセンサとすることができ、酸化還元酵素として乳酸デヒドロゲナーゼを用いる場合は乳酸センサとすることができる。 <Biosensor>
Mutant cytochrome proteins of the present invention can be used in electrochemical biosensors that utilize redox reactions. The type of biosensor can be used for various purposes depending on the type of oxidoreductase to be combined. For example, when glucose oxidoreductase is used as the oxidoreductase, it can be used as a glucose sensor, and lactate dehydrogenase can be used as the oxidoreductase. When used, it can be a lactate sensor.

この中ではグルコースセンサが好ましく、グルコースセンサを作製する場合、酵素電極の試薬層に本発明の変異型シトクロムタンパク質と酸化還元酵素の触媒サブユニットを含有させることが好ましい。さらにγサブユニットを含有させてもよい。これにより、試料中のグルコースを触媒サブユニットにより酸化し、このとき生じた電子を変異型シトクロムタンパク質が受容し、電極に伝達することで、試料中のグルコース濃度に応じた応答電流が流れ、その電流値によりグルコース濃度を算出することができる。 Among these, a glucose sensor is preferable, and when a glucose sensor is produced, it is preferable to incorporate the mutant cytochrome protein of the present invention and the catalytic subunit of an oxidoreductase into the reagent layer of the enzyme electrode. A γ subunit may also be included. As a result, the glucose in the sample is oxidized by the catalytic subunit, and the electrons generated at this time are received by the mutant cytochrome protein and transferred to the electrode, resulting in a response current corresponding to the glucose concentration in the sample. The glucose concentration can be calculated from the current value.

グルコースセンサとして具体的には、作用極として、本発明の変異型シトクロムタンパク質とグルコースデヒドロゲナーゼの触媒サブユニットを含む複合体が金電極、白金電極、カーボン電極等の電極表面上に固定化された酵素電極を用いるグルコースセンサが挙げられる。センサは、目的とする被検物質の濃度を電気化学的に測定する測定系をいうが、作用極（酵素電極）、対極（白金等）、および参照極（Ａｇ／ＡｇＣｌ等）の３電極を含む計が好ましい。慣用の簡易血糖値システムにおいて用いられているような、作用極と対極とから構成される２電極系でもよい。センサはさらに、緩衝液および被検試料を入れる恒温セル、作用極に電位を印加する電源、電流計、記録計等を含むことが好ましい。センサは、バッチ型であってもフロー型であってもよい。特にフロー型のセンサとしては、血糖値を連続で計測できるセンサであってもよい。すなわち、連続的に供給される血液試料、あるいは同透析試料、あるいは血液中あるいは細胞間質液中に本発明の酵素を固定した二
電極系あるいは三電極系を挿入して計測するセンサであってもよい。このような酵素センサの構造は、当該技術分野においてよく知られており、例えばＢｉｏｓｅｎｓｏｒｓ－Ｆｕｎｄａｍｅｎｔａｌ ａｎｄ Ａｐｐｌｉｃａｔｉｏｎｓ－Ａｎｔｈｏｎｙ Ｐ．Ｆ．Ｔｕｒｎｅｒ，Ｉｓａｏ Ｋａｒｕｂｅ ａｎｄ Ｇｅｒｏｇｅ Ｓ． Ｗｉｌｓｏｎ，Ｏｘｆｏｒｄ Ｕｎｉｖｅｒｓｉｔｙ Ｐｒｅｓｓ １９８７に記載されている。 Specifically, as a glucose sensor, an enzyme in which a complex containing the mutant cytochrome protein of the present invention and a catalytic subunit of glucose dehydrogenase is immobilized on the surface of an electrode such as a gold electrode, a platinum electrode, or a carbon electrode as a working electrode. Glucose sensors using electrodes are included. A sensor refers to a measurement system that electrochemically measures the concentration of a target substance to be tested. An inclusive total is preferred. A two-electrode system composed of a working electrode and a counter electrode, such as those used in conventional simple blood glucose level systems, may also be used. Preferably, the sensor further includes a thermostatic cell containing a buffer solution and a sample to be tested, a power supply for applying a potential to the working electrode, an ammeter, a recorder, and the like. The sensor may be batch or flow type. In particular, the flow-type sensor may be a sensor capable of continuously measuring the blood sugar level. That is, a sensor that performs measurement by inserting a two-electrode system or a three-electrode system in which the enzyme of the present invention is immobilized in continuously supplied blood samples, dialyzed samples of the same, blood or interstitial fluid. good too. The construction of such enzymatic sensors is well known in the art, see, for example, Biosensors-Fundamental and Applications-Anthony P.; F. Turner, Isao Karube and George S.; Wilson, Oxford University Press 1987.

本発明のグルコースセンサを用いるグルコースの濃度の測定は、例えば、以下のようにして行うことができる。センサの恒温セルに緩衝液を入れ、一定温度に維持する。作用電極として本発明の変異型シトクロムタンパク質とグルコース酸化還元酵素の触媒サブユニットタンパク質を固定化した酵素電極を用い、対極としては例えば白金電極を、参照電極としては例えばＡｇ／ＡｇＣｌ電極を用いる。作用極に一定の電位（例えば、銀・塩化銀電極に対して０～＋３００ｍＶ、好ましくは０～＋１５０ｍＶ、より好ましくは０～＋１００ｍＶ）を印加して、電流が定常になった後、恒温セルにグルコースを含む試料を加えて電流の増加を測定する。標準濃度のグルコース溶液により作製したキャリブレーションカーブに従い、試料中のグルコース濃度を計算することができる。 Measurement of glucose concentration using the glucose sensor of the present invention can be performed, for example, as follows. The thermostatic cell of the sensor is filled with buffer and maintained at a constant temperature. An enzyme electrode in which the mutant cytochrome protein of the present invention and the catalytic subunit protein of glucose oxidoreductase are immobilized is used as the working electrode, a platinum electrode is used as the counter electrode, and an Ag/AgCl electrode is used as the reference electrode. After applying a constant potential to the working electrode (for example, 0 to +300 mV, preferably 0 to +150 mV, more preferably 0 to +100 mV with respect to the silver/silver chloride electrode) and the current becomes steady, the A sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to the calibration curve generated with standard concentration glucose solutions.

本発明の変異型シトクロムタンパク質とＧＤＨ触媒サブユニットを含むＧＤＨ複合体は、グルコースアッセイキットの構成要素として用いることもできる。グルコースアッセイキットには、本発明の変異型シトクロムタンパク質とＧＤＨ触媒サブユニットを含むＧＤＨ複合体以外に、発色又は発光試薬、希釈用緩衝液、標準物質、使用説明書などが含まれてよい。 A GDH complex comprising a mutant cytochrome protein of the invention and a GDH catalytic subunit can also be used as a component of a glucose assay kit. The glucose assay kit may contain a chromogenic or luminescent reagent, a dilution buffer, standards, instructions, etc., in addition to the mutant cytochrome protein of the present invention and a GDH complex comprising a GDH catalytic subunit.

例えば、ブルクホルデリア・セパシアの野生型ＧＤＨを用いたグルコースセンサ及びグルコースアッセイキットは、米国特許公開第２００４／００２３３３０Ａ１に記載されている。本発明の変異型シトクロムタンパク質を含むＧＤＨも、同様にして使用することができる。 For example, a glucose sensor and glucose assay kit using Burkholderia cepacia wild-type GDH is described in US Patent Publication No. 2004/0023330A1. GDHs containing mutant cytochrome proteins of the invention can be used in a similar manner.

次に、実施例を挙げて本発明をさらに具体的に説明するが、本発明はこれら実施例に何ら限定されるものではない。 EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

〔実施例１〕変異型ＧＤＨ βサブユニット遺伝子の構築
変異型ＧＤＨ βサブユニット遺伝子の構築に用いるプラスミドとしては、特開２０１２
－０９０５６３に記載のプラスミドｐＴｒｃ９９Ａγαβにおいて、αサブユニットに変異を導入したｐＴｒｃ９９Ａγα（ＱＹＹ）βを使用した。同プラスミドは、ブルクホルデリア・セパシアＫＳ１株（ＦＥＲＭ ＢＰ－７３０６）染色体ＤＮＡから単離された、ＧＤＨ γサブユニット構造遺伝子とαサブユニット構造遺伝子、βサブユニット構造遺伝子を連続して含み、このうちαサブユニット構造遺伝子について、３２６残基目のセリン、３６５残基目のセリン、４７２残基目のアラニンをコードするコドンがそれぞれグルタミン、チロシン、チロシンをコードするものに置換されたＤＮＡ断片が、ベクターｐＴｒｃ９９Ａのクローニング部位に挿入されてなるプラスミドである。本プラスミド中のＧＤＨの各構造遺伝子は、ｔｒｃプロモーターによって制御される。ｐＴｒｃ９９Ａγαβは、アンピシリン耐性遺伝子を保持している。 [Example 1] Construction of mutant GDH β subunit gene As a plasmid used for constructing the mutant GDH β subunit gene, JP-A-2012
In the plasmid pTrc99Aγαβ described in -090563, pTrc99Aγα(QYY)β in which the α subunit was mutated was used. The plasmid contains the GDH γ-subunit structural gene, the α-subunit structural gene, and the β-subunit structural gene, isolated from the chromosomal DNA of Burkholderia cepacia KS1 (FERM BP-7306). Among them, regarding the α subunit structural gene, a DNA fragment in which the codons encoding serine at residue 326, serine at residue 365, and alanine at residue 472 were replaced with those encoding glutamine, tyrosine, and tyrosine, respectively. , which is inserted into the cloning site of the vector pTrc99A. Each structural gene of GDH in this plasmid is controlled by the trc promoter. pTrc99Aγαβ carries the ampicillin resistance gene.

まず、上記プラスミドＤＮＡを鋳型として、以下の配列を有するオリゴヌクレオチドをプライマーとするＰＣＲにより、ＧＤＨのγサブユニット、αサブユニット及びβサブユニットのシグナル配列までをコードするＤＮＡ断片を増幅した。 First, using the above plasmid DNA as a template, a DNA fragment encoding up to the signal sequences of the γ subunit, α subunit and β subunit of GDH was amplified by PCR using oligonucleotides having the following sequences as primers.

〔フォワードプライマー〕
５’－ＡＣＧＴＡＧＣＣＡＴＧＧＣＡＣＡＣＡＡＣＧＡＣＡＡＣＣＡＣＴＣ－３’（配列
番号１４）
〔リバースプライマー〕
５’－ＡＴＣＧＧＣＣＧＣＧＣＧＣＧＣＧＡＡＧＣＣＣＧＧＣＡＡ－３’（配列番号１５） [Forward primer]
5′-ACGTAGCCATGGCACACAACGACAACCACTC-3′ (SEQ ID NO: 14)
[Reverse primer]
5′-ATCGGCCGCGCGCGCGAAGCCCGGGCAA-3′ (SEQ ID NO: 15)

ＰＣＲ反応は、以下の反応組成で、９５℃、３０秒の後、９５℃ ３０秒、５５℃ １分、７２℃ ２分を２５サイクル繰り返し、７２℃ ５分の反応を行った後、４℃で保持した。 The PCR reaction has the following reaction composition, and after 95°C for 30 seconds, repeats 25 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 2 minutes. held with

〔反応液組成〕
ｐＴｒｃ９９Ａγα（ＱＹＹ）β（１００ｎｇ／μｌ） ０．５μｌ
１０×反応緩衝液 ２μｌ
フォワードプライマー（１００ｎｇ／μｌ） ０．５μｌ
リバースプライマー（１００ｎｇ／μｌ ） ０．５μｌ
ｄＮＴＰ ０．４μｌ
蒸留水 １５．７μｌ
ＤＮＡポリメラーゼ ０．４μｌ
合計 ２０μｌ [Reaction liquid composition]
pTrc99Aγα(QYY)β (100 ng/μl) 0.5 μl
2 μl of 10× reaction buffer
Forward primer (100 ng/μl) 0.5 μl
Reverse primer (100 ng/μl) 0.5 μl
0.4 μl of dNTPs
Distilled water 15.7 μl
DNA polymerase 0.4 μl
Total 20 μl

また、βサブユニットの第３ヘム結合ドメイン、すなわち配列番号４のアミノ酸番号３１４～４２５または３３０～４２５の領域をコードするＤＮＡ断片を、以下の配列を有するオリゴヌクレオチドをプライマーとするＰＣＲにより増幅した。なお、フォワードプライマーにはどちらも５‘末端側にβサブユニットのシグナル配列に相当する配列が付加されている。 In addition, a DNA fragment encoding the third heme-binding domain of the β subunit, that is, the region of amino acid numbers 314 to 425 or 330 to 425 of SEQ ID NO:4 was amplified by PCR using oligonucleotides having the following sequences as primers. . Both forward primers have a sequence corresponding to the signal sequence of the β subunit added to the 5' end.

〔フォワードプライマー ３１４～４２５増幅用〕
５’－ＴＴＧＣＣＧＧＧＣＴＴＣＧＣＧＣＧＣＧＣＧＧＣＣＧＡＴＣＴＧＣＧＣＧＧＴＧＴＣＧＣＧＣＴＣＧＣＧ－３’（配列番号１６）
〔フォワードプライマー ３３０～４２５増幅用〕
５’－ＴＴＧＣＣＧＧＧＣＴＴＣＧＣＧＣＧＣＧＣＧＧＣＣＧＡＴＴＡＴＣＴＣＧＧＣＡＡＣＴＧＣＧＣＧＡＣＧ－３’（配列番号１７）
〔リバースプライマー〕
５’－ＧＴＧＧＴＧＣＴＣＧＡＧＴＧＣＧＧＣＣＧＣＧＣＧＣＡＧＣＴＴＣＧＣＧＡＣＧＴＣＣＴＧ－３’（配列番号１８） [Forward primer 314-425 for amplification]
5′-TTGCCGGGCTTCGCGCGCGCGGCCGATCTGCGCGGTGTCGCGCTCGCG-3′ (SEQ ID NO: 16)
[Forward primer 330-425 for amplification]
5′-TTGCCGGGCTTCGCGCGCGCGGCCGATTATCTCGGCCAACTGCGCGACG-3′ (SEQ ID NO: 17)
[Reverse primer]
5′-GTGGTGCTCGAGTGCGGCCGCGCGCAGCTTCGCGACGTCCTG-3′ (SEQ ID NO: 18)

ＰＣＲ反応は、以下の反応組成で、９５℃、３０秒の後、９５℃ ３０秒、５５℃ １分、７２℃ ３０秒を２５サイクル繰り返し、７２℃ ５分の反応を行った後、４℃で保持した。 The PCR reaction has the following reaction composition, and after 95°C for 30 seconds, repeats 25 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 30 seconds. held with

〔反応液組成〕
ｐＴｒｃ９９Ａγα（ＱＹＹ）β（１００ｎｇ／μｌ） ０．５μｌ
１０×反応緩衝液 ２μｌ
フォワードプライマー（１００ｎｇ／μｌ） ０．５μｌ
リバースプライマー（１００ｎｇ／μｌ ） ０．５μｌ
ｄＮＴＰ ０．４μｌ
蒸留水 １５．７μｌ
ＤＮＡポリメラーゼ ０．４μｌ
合計 ２０μｌ [Reaction liquid composition]
pTrc99Aγα(QYY)β (100 ng/μl) 0.5 μl
2 μl of 10× reaction buffer
Forward primer (100 ng/μl) 0.5 μl
Reverse primer (100 ng/μl) 0.5 μl
0.4 μl of dNTPs
Distilled water 15.7 μl
DNA polymerase 0.4 μl
Total 20 μl

上記操作で得られたＤＮＡ断片をそれぞれ精製したのち、これらを混合して鋳型とし、以
下の配列を有するオリゴヌクレオチドをプライマーとするＯｖｅｒｌａｐ ｅｘｔｅｎｔｉｏｎ ＰＣＲにより結合、増幅した。なお、リバースプライマーにはヒスチジンタグの配列がデザインされており、これにより変異型βサブユニットのＣ末端にヒスチジンタグが付加されることとなる。 After purifying each of the DNA fragments obtained by the above operation, they were mixed and used as a template, and bound and amplified by overlap extension PCR using oligonucleotides having the following sequences as primers. A histidine tag sequence was designed in the reverse primer, which adds a histidine tag to the C-terminus of the mutant β subunit.

〔フォワードプライマー〕
５’－ＡＣＧＴＡＧＣＣＡＴＧＧＣＡＣＡＣＡＡＣＧＡＣＡＡＣＣＡＣＴＣ－３’（配列番号１４）
〔リバースプライマー〕
５’－ＧＴＡＣＧＴＡＡＧＣＴＴＴＣＡＧＴＧＧＴＧＧＴＧＧＴＧＧＴＧＧＴＧＣＴＣＧＡＧＴＧＣＧＧＣＣＧＣ－３’（配列番号１９） [Forward primer]
5′-ACGTAGCCATGGCACACAACGACAACCACTC-3′ (SEQ ID NO: 14)
[Reverse primer]
5′-GTACGTAAGCTTTTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGC-3′ (SEQ ID NO: 19)

ＰＣＲ反応は、以下の反応組成で、９５℃、３０秒の後、９５℃ ３０秒、５５℃ １分、７２℃ ４分を２５サイクル繰り返し、７２℃ ５分の反応を行った後、４℃で保持した。 The PCR reaction had the following reaction composition, and after 95°C for 30 seconds, 25 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 4 minutes were repeated. held with

〔反応液組成〕
γサブユニット～βサブユニットシグナル配列までのＰＣＲ産物（１００ｎｇ／μｌ）
１μｌ
βサブユニット ３１４～４２５までのＰＣＲ産物
またはβサブユニット ３３０～４２５までのＰＣＲ産物（１００ｎｇ／μｌ）
１μｌ
１０×反応緩衝液 ２μｌ
フォワードプライマー（１００ｎｇ／μｌ） ０．５μｌ
リバースプライマー（１００ｎｇ／μｌ ） ０．５μｌ
ｄＮＴＰ ０．４μｌ
蒸留水 １４．２μｌ
ＤＮＡポリメラーゼ ０．４μｌ
合計 ２０μｌ [Reaction liquid composition]
PCR product from γ subunit to β subunit signal sequence (100 ng/μl)
1 μl
PCR product from β subunit 314 to 425 or PCR product from β subunit 330 to 425 (100 ng/μl)
1 μl
2 μl of 10× reaction buffer
Forward primer (100 ng/μl) 0.5 μl
Reverse primer (100 ng/μl) 0.5 μl
0.4 μl of dNTPs
Distilled water 14.2 μl
DNA polymerase 0.4 μl
Total 20 μl

得られたＰＣＲ産物を精製後、Ｎ末端側をＮｃｏＩ、Ｃ末端側をＨｉｎｄＩＩＩで消化し、同様に処理したｐＴｒｃ９９Ａにライゲーションした。得られた組換えベクターでエシェリヒア・コリＤＨ５αを形質転換し、カルベニシリン５０μｇ／ｍＬを含むＬＢ寒天培地で生じるコロニーを採取した。得られた形質転換体を液体のＬＢ培地で培養してプラスミドを抽出し、その挿入ＤＮＡ断片を解析したところ、γサブユニット、αサブユニット、βサブユニットのシグナル配列に続き、βサブユニットの３１４～４２５残基目をコードする遺伝子または同３３０～４２５残基目をコードする遺伝子が挿入されていることが確認された。これらのプラスミドを、それぞれｐＴｒｃ９９Ａγα（ＱＹＹ）β３１４－Ｈｉｓ、ｐＴｒｃ９９Ａγα（ＱＹＹ）β３３０－Ｈｉｓと命名した。これらのプラスミド中のＧＤＨの各構造遺伝子は、βサブユニットのＣ末端にヒスチジンタグを有し、ｔｒｃプロモーターによって制御される。また、いずれもアンピシリン耐性遺伝子を保持している。 After purification of the resulting PCR product, the N-terminal side was digested with NcoI and the C-terminal side with HindIII, and ligated to similarly treated pTrc99A. Escherichia coli DH5α was transformed with the resulting recombinant vector, and colonies formed on LB agar medium containing 50 μg/mL carbenicillin were collected. The resulting transformant was cultured in liquid LB medium to extract the plasmid, and the inserted DNA fragment was analyzed. It was confirmed that the gene encoding the 314th to 425th residues or the gene encoding the 330th to 425th residues were inserted. These plasmids were named pTrc99Aγα(QYY)β314-His and pTrc99Aγα(QYY)β330-His, respectively. Each GDH structural gene in these plasmids has a histidine tag at the C-terminus of the β subunit and is controlled by the trc promoter. In addition, both have ampicillin resistance genes.

〔実施例２〕変異型ＧＤＨ βサブユニットを含むブルクホルデリア・セパシアのＧＤＨ
の発現
実施例１で得られた発現プラスミドを用いて、変異型ＧＤＨ βサブユニット遺伝子を含
むＧＤＨを製造した。 [Example 2] Burkholderia cepacia GDH containing mutant GDH β subunit
Using the expression plasmid obtained in Example 1, GDH containing the mutant GDH β subunit gene was produced.

変異型ＧＤＨ βサブユニット遺伝子を含むＧＤＨ発現プラスミドおよびシトクロムｃ成
熟に必須の遺伝子群を有するプラスミドｐＢＢＪＭｃｃｍを導入したエシェリヒア・コリ
ＢＬ２１（ＤＥ３）株を、３ｍｌのＬＢ培地（カルベニシリン５０μｇ／ｍｌ及びカナマイシン５０μｇ／ｍｌ含有）で、試験管を用いて３７℃で一晩振とう培養した。それらの培養液を、培養液１リットル当たりの組成が表１となるように調製された培地５０ｍｌ（カルベニシリン５０μｇ／ｍｌおよびカナマイシン５０μｇ／ｍｌ含有）を含む３００ｍｌの坂口フラスコに２本に１％植菌し、２５℃で２８時間振とう培養した。なお、宿主が恒常的にシトクロムｃを成熟させることができる場合、例えば、ｃｃｍ遺伝子がゲノムに相同的組み換えなどで挿入され、恒常的に発現している形質転換株などでは、ｐＢＢＪＭｃｃｍのようなベクタは不要である。 The Escherichia coli BL21 (DE3) strain introduced with the GDH expression plasmid containing the mutant GDH β subunit gene and the plasmid pBBJMccm having the gene cluster essential for cytochrome c maturation was added to 3 ml of LB medium (50 μg/ml carbenicillin and 50 μg kanamycin). /ml) and shake-cultured overnight at 37°C using a test tube. Two 300 ml Sakaguchi flasks containing 50 ml of medium (containing 50 µg/ml of carbenicillin and 50 µg/ml of kanamycin) prepared so that the composition per liter of culture solution is as shown in Table 1 were used for 1% seeding. It was cultured with shaking at 25° C. for 28 hours. In addition, when the host can constantly mature cytochrome c, for example, in a transformant strain in which the ccm gene is inserted into the genome by homologous recombination or the like and is constantly expressed, a vector such as pBBJMccm is unnecessary.

Figure 0007339723000001
Figure 0007339723000001

前記で培養した培養液から菌体を集め、得られた湿菌体１ｇあたり５ｍｌのＢｕｇＢｕｓｔｅｒ Ｐｒｏｔｅｉｎ Ｅｘｔｒａｃｔｉｏｎ ｒｅａｇｅｎｔ（メルク・ミリポア）を添加、懸濁し、菌体を溶解させた。この懸濁液を遠心分離（１５０００ｒ．ｐ．ｍ、２０分、４℃）して残渣を除去し、粗酵素サンプルとした。 Cells were collected from the culture broth cultured above, and 5 ml of BugBuster Protein Extraction reagent (Merck Millipore) was added to 1 g of the obtained wet cells and suspended to dissolve the cells. This suspension was centrifuged (15000 rpm, 20 minutes, 4° C.) to remove the residue and used as a crude enzyme sample.

また、γα（ＱＹＹ）β３１４－Ｈｉｓについては、粗酵素サンプルを０．５Ｍ塩化ナトリウム、２０ｍＭイミダゾールを含む２０ｍＭリン酸ナトリウムバッファー（ｐＨ７．０）に対して一晩透析したのち、同バッファーで平衡化したＮｉ－ＮＴＡアガロース（キアゲン）を充てんしたカラムに添加し、０．５Ｍ塩化ナトリウム、５４ｍＭイミダゾールを含む２０ｍＭリン酸ナトリウムバッファー（ｐＨ７．０）で洗浄した後、０．５Ｍ塩化ナトリウム、１４０ｍＭイミダゾールを含む２０ｍＭリン酸ナトリウムバッファー（ｐＨ７．０）で溶出させ、精製酵素サンプルを得た。 For γα(QYY)β314-His, the crude enzyme sample was dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M sodium chloride and 20 mM imidazole, and then equilibrated with the same buffer. After adding to a column packed with Ni-NTA agarose (Qiagen) and washing with 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 M sodium chloride and 54 mM imidazole, 0.5 M sodium chloride and 140 mM imidazole were added. A purified enzyme sample was obtained by elution with 20 mM sodium phosphate buffer (pH 7.0).

〔実施例３〕変異型ＧＤＨ βサブユニットを含むブルクホルデリア・セパシアのＧＤＨ
の機能解析
ＧＤＨ活性の測定は、ＰＭＳ／ＤＣＩＰ系およびルテニウム（Ｒｕ）／ＭＴＴ系で行った。前者は一般的なＧＤＨ活性を、後者はβサブユニットを介してメディエータへと電子伝達が行われた場合のＧＤＨ活性を示す。 [Example 3] Burkholderia cepacia GDH containing mutant GDH β subunit
Functional analysis of GDH activity was performed with the PMS/DCIP system and the ruthenium (Ru)/MTT system. The former shows general GDH activity, and the latter shows GDH activity when electrons are transferred to the mediator via the β subunit.

ＰＭＳ／ＤＣＩＰ系での活性測定は以下のように行った。２０ｍＭリン酸カリウムバッファー（ｐＨ７．０）中で、酵素サンプル、終濃度０．６ｍＭのメチルフェナジンメトサルフェート（ＰＭＳ）、終濃度０．０６ｍＭの２，６－ジクロロフェノールインドフェノール（ＤＣＩＰ）、終濃度４ｍＭまたは４０ｍＭのグルコースを添加し、分光光度計を用いてＤＣＩＰ由来の吸収波長である６００ｎｍの吸光度の１分間当たりの変化量を測定した
。
また、Ｒｕ／ＭＴＴ系での活性測定は以下のように行った。２０ｍＭリン酸カリウムバッファー（ｐＨ７．０）中で、酵素サンプル、終濃度２％のヘキサアンミンルテニウム（ＩＩＩ）塩化物、終濃度１ｍＭの３－（４，５－ジメチル－２－チアゾリル）－２，５－ジフェニル－２Ｈ－テトラゾリウムブロミド（ＭＴＴ）、終濃度４ｍＭまたは４０ｍＭのグルコースを添加し、分光光度計を用いてＭＴＴから生じるホルマザン由来の吸収波長である５６５ｎｍの吸光度の１分間当たりの変化量を測定した。
粗酵素サンプルの活性測定の結果を、表２に示す。 Activity measurement in the PMS/DCIP system was performed as follows. Enzyme sample, 0.6 mM final concentration methylphenazine methosulfate (PMS), 0.06 mM final concentration 2,6-dichlorophenolindophenol (DCIP), final concentration in 20 mM potassium phosphate buffer (pH 7.0) 4 mM or 40 mM glucose was added, and the amount of change per minute in absorbance at 600 nm, which is the absorption wavelength derived from DCIP, was measured using a spectrophotometer.
Further, activity measurement in the Ru/MTT system was performed as follows. Enzyme sample, hexaammineruthenium (III) chloride at a final concentration of 2%, 3-(4,5-dimethyl-2-thiazolyl)-2 at a final concentration of 1 mM, in 20 mM potassium phosphate buffer (pH 7.0). 5-Diphenyl-2H-tetrazolium bromide (MTT) was added to a final concentration of 4 mM or 40 mM glucose, and the amount of change in absorbance per minute at 565 nm, which is the absorption wavelength derived from formazan generated from MTT using a spectrophotometer, was measured. It was measured.
Table 2 shows the results of the activity measurement of the crude enzyme samples.

Figure 0007339723000002
Figure 0007339723000002

表中、野生型βサブユニットを含む酵素には及ばないものの、両変異体ともＲｕ／ＭＴＴ系でＧＤＨ活性を示しており、これらの変異体がβサブユニットを介してメディエータへの電子伝達を行うことが示唆された。また、野生型酵素ではβサブユニットを含まないγα複合体ではＰＭＳ／ＤＣＩＰを電子受容体としたときには酵素活性は約45U/mgであり（Biochimica et Biophysica Acta, 1645(2), 133-138.）、βサブユニットを含む場合の値（約300U/mg：Journal of Biotechnology, 123(2), 127-136.）の約１５％の酵素活性し
か観察されないが、本変異シトクロムｃを含む複合体は野生型βサブユニット複合体を含む場合とほとんど同じ活性を示した。したがって、この変異βサブユニットがαサブユニットから電子を正常に受け取り、これをPMS/DCIPに電子を受け渡していることが見出された。 In the table, both mutants exhibited GDH activity in the Ru/MTT system, though not as high as the enzyme containing the wild-type β subunit. suggested to do so. In addition, the enzyme activity of the γα complex that does not contain the β subunit in the wild-type enzyme is about 45 U/mg when PMS/DCIP is used as the electron acceptor (Biochimica et Biophysica Acta, 1645(2), 133-138. ), the value when containing the β subunit (about 300 U / mg: Journal of Biotechnology, 123 (2), 127-136.) Only about 15% of the enzyme activity is observed, but the complex containing this mutant cytochrome c showed almost the same activity as that containing the wild-type β-subunit complex. Therefore, it was found that this mutant β subunit normally receives electrons from the α subunit and transfers them to PMS/DCIP.

また、γα（ＱＹＹ）β３１４について精製を行い、上記手順に従って４０ｍＭグルコースに対する酵素活性を、別途調製した野生型βサブユニットを含むγα（ＱＹＹ）βともども測定した。結果を表３に示す。 γα(QYY)β314 was also purified, and the enzymatic activity against 40 mM glucose was measured according to the procedure described above together with γα(QYY)β containing the wild-type β subunit prepared separately. Table 3 shows the results.

Figure 0007339723000003
Figure 0007339723000003

精製酵素においてもγα（ＱＹＹ）β３１４はＲｕ／ＭＴＴ系でＧＤＨ活性を示しており、ＰＭＳ／ＤＣＩＰ系でのＧＤＨ活性との比は野生型βサブユニットを含む酵素複合体と同様であった。このことから、当該変異体が野生型βサブユニットを含む酵素複合体と同様、βサブユニットを介してメディエータへの電子伝達を行うことが示唆された。 In the purified enzyme, γα(QYY)β314 also exhibited GDH activity in the Ru/MTT system, and the ratio to the GDH activity in the PMS/DCIP system was similar to that of the enzyme complex containing the wild-type β subunit. This suggests that the mutant transfers electrons to the mediator via the β subunit, similar to the enzyme complex containing the wild-type β subunit.

〔実施例４〕グルコースセンサの作製
金表面に単分子膜形成分子を介して変異型βサブユニットを含むGDHを固定化した酵素電
極を作製した。単分子膜形成分子としては、以下のDSHを用いた。

Figure 0007339723000004
[Example 4] Preparation of glucose sensor An enzyme electrode was prepared by immobilizing GDH containing a mutant β subunit on a gold surface via a monolayer-forming molecule. The following DSH was used as a monolayer-forming molecule.
Figure 0007339723000004

具体的には、金ワイヤ（径０．５ｍｍ、長さ６～７ｃｍ）をピランハ溶液（200μl）に室温で２時間浸漬し、その後、アセトンで洗浄し、DSHのアセトン溶液（濃度20μM）に浸漬し、２５℃で２４時間インキュベートしてDSHのチオール基を金表面に結合させた。続い
て、アセトンで洗浄し、上記ＧＤＨα、βサブユニット複合体(濃度0.03mg/ml)）を含む
リン酸バッファー（300μl）に浸漬し、４℃で一晩インキュベートしてDSHの官能基を介
して酵素複合体を結合させ、酵素電極を得た。 Specifically, a gold wire (diameter 0.5 mm, length 6-7 cm) was immersed in a Piranha solution (200 μl) for 2 hours at room temperature, then washed with acetone and immersed in an acetone solution of DSH (concentration 20 μM). and incubated at 25° C. for 24 hours to bind the thiol groups of DSH to the gold surface. Subsequently, it was washed with acetone, immersed in phosphate buffer (300 µl) containing the above-mentioned GDHα, β subunit complex (concentration 0.03 mg/ml), and incubated overnight at 4°C to allow the DSH functional groups to pass through. An enzyme electrode was obtained by binding the enzyme complex with the

〔実施例５〕グルコース濃度の測定
上記酵素電極を用いて、0mM（バックグラウンド）、1mM、 5mMまたは50mMのグルコース水溶液に対する応答電流値の測定をクロノアンペアメトリーにより行った。グルコース測定は、対極（Ptワイヤ）、参照極（銀/塩化銀）を用い、作用極への印加電位を０Ｖ、＋０
．１Ｖ、＋０．４Ｖ（vs.銀/塩化銀）とし、３７℃で行った。 [Example 5] Measurement of glucose concentration Using the enzyme electrode described above, response current values to 0 mM (background), 1 mM, 5 mM or 50 mM glucose aqueous solutions were measured by chronoamperemetry. For glucose measurement, a counter electrode (Pt wire) and a reference electrode (silver/silver chloride) are used, and the applied potential to the working electrode is 0 V, +0
. 1 V, +0.4 V (vs. silver/silver chloride) and carried out at 37°C.

＜結果＞
銀塩化銀電極に対して400mV印加した電流値のグルコース濃度特性を基準に、0mVおよび100mVの電流値を比較した。
その結果、図１に示されるように、印加電位0mVにおいては比較例（点線）でグルコース
濃度依存的な酸化電流が観測されなかったのに対し、実施例（実線）では酸化電流のグル
コース濃度依存が見られた。
印加電位100mVで比較例でもグルコース濃度依存的な酸化電流の観測がされたが、400mVの応答電流を基準とした電流値出力は実施例の方が高かった。
以上から実施例のtruncated βサブユニットを用いたセンサにおいて、野生型βサブユニットを用いたセンサより低い酸化電位で触媒電流を取得可能であることが示された。 <Results>
Current values of 0 mV and 100 mV were compared based on the glucose concentration characteristics of a current value of 400 mV applied to a silver-silver chloride electrode.
As a result, as shown in FIG. 1, at an applied potential of 0 mV, no glucose concentration-dependent oxidation current was observed in the comparative example (dotted line), whereas in the example (solid line), the glucose concentration-dependent oxidation current was observed. It was observed.
At an applied potential of 100 mV, a glucose concentration-dependent oxidation current was also observed in the comparative example, but the current value output based on the response current of 400 mV was higher in the example.
From the above, it was shown that the sensor using the truncated β subunit of the example can obtain a catalytic current at a lower oxidation potential than the sensor using the wild-type β subunit.

本発明の変異型シトクロムタンパク質を用いたバイオセンサは、低電位での電流測定が可能であり、グルコースセンサ等のバイオセンサに好適に使用することができる。 A biosensor using the mutant cytochrome protein of the present invention is capable of current measurement at a low potential, and can be suitably used as a biosensor such as a glucose sensor.

Claims (16)

配列番号４のアミノ酸配列または配列番号４と６０％以上のアミノ酸配列同一性を有するアミノ酸配列からなり、３つのヘム結合ドメインを有する、シトクロムＣタンパク質において、Ｎ末端側から数えて１番目の第１ヘム結合ドメインおよび２番目の第２ヘム結合ドメインが欠損した、変異型シトクロムＣタンパク質。 The cytochrome C protein consisting of the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having an amino acid sequence identity of 60% or more with SEQ ID NO: 4 and having three heme-binding domains , counted from the N-terminal side, the first A mutant cytochrome C protein lacking the heme-binding domain and the second second heme-binding domain. 前記第１および第２ヘム結合ドメインを含む領域が欠損した、請求項１に記載の変異型シトクロムＣタンパク質。 2. The mutant cytochrome C protein of claim 1, wherein the region containing said first and second heme-binding domains is deleted. 前記第１および第２ヘム結合ドメインを含む領域が配列番号４の４３～１９５の領域に相当する、請求項２に記載の変異型シトクロムＣタンパク質。 3. The mutant cytochrome C protein of claim 2, wherein the region containing said first and second heme-binding domains corresponds to region 43-195 of SEQ ID NO:4. シトクロムＣタンパク質がブルクホルデリア属微生物に由来する、請求項１～３のいずれか一項に記載の変異型シトクロムＣタンパク質。 The mutant cytochrome C protein according to any one of claims 1 to 3 , wherein the cytochrome C protein is derived from a Burkholderia microorganism. シトクロムＣタンパク質がブルクホルデリア・セパシアに由来する、請求項４に記載の変異型シトクロムＣタンパク質。 5. The mutant cytochrome C protein of claim 4 , wherein the cytochrome C protein is derived from Burkholderia cepacia. 改変前のシトクロムＣタンパク質が、配列番号４と８０％以上のアミノ酸配列同一性を有する、請求項１～５のいずれか一項に記載の変異型シトクロムＣタンパク質。The mutant cytochrome C protein according to any one of claims 1 to 5, wherein the cytochrome C protein before modification has 80% or more amino acid sequence identity with SEQ ID NO:4. 改変前のシトクロムＣタンパク質が、配列番号４と９０％以上のアミノ酸配列同一性を有する、請求項１～６のいずれか一項に記載の変異型シトクロムＣタンパク質。 The mutant cytochrome C protein according to any one of claims 1 to 6, wherein the cytochrome C protein before modification has 90 % or more amino acid sequence identity with SEQ ID NO:4. 配列番号４のアミノ酸番号３１４～４２５のアミノ酸配列、配列番号４のアミノ酸番号３３０～４２５のアミノ酸配列、またはこれらのアミノ酸配列において１または数個のアミノ酸が置換、欠失、挿入もしくは付加されたアミノ酸配列（ただし、アミノ酸番号３３４～３３８のＣＸＸＣＨモチーフは改変されない）からなる、請求項１～７のいずれか一項
に記載の変異型シトクロムＣタンパク質。 Amino acid sequences of amino acid numbers 314 to 425 of SEQ ID NO: 4, amino acid sequences of amino acid numbers 330 to 425 of SEQ ID NO: 4, or amino acids in which one or several amino acids are substituted, deleted, inserted or added in these amino acid sequences 8. A mutant cytochrome C protein according to any one of claims 1 to 7, consisting of the sequence, provided that the CXXCH motif of amino acid numbers 334-338 is not modified. 請求項１～８のいずれか一項に記載の変異型シトクロムＣタンパク質をコードするＤＮＡ。 A DNA encoding the mutant cytochrome C protein according to any one of claims 1 to 8. 請求項９に記載のＤＮＡを含む組み換えベクター。 A recombinant vector comprising the DNA of claim 9 . 請求項１０に記載の組み換えベクターで形質転換された形質転換細胞。 A transformed cell transformed with the recombinant vector according to claim 10 . 請求項１～８のいずれか一項に記載の変異型シトクロムＣタンパク質および酸化還元酵素の触媒サブユニットタンパク質を含む、酸化還元酵素・シトクロム複合体。 An oxidoreductase/cytochrome complex comprising the mutant cytochrome C protein according to any one of claims 1 to 8 and an oxidoreductase catalytic subunit protein. 酸化還元酵素がグルコースデヒドロゲナーゼである、請求項１２に記載の酸化還元酵素・シトクロム複合体。 13. The oxidoreductase-cytochrome complex according to claim 12, wherein the oxidoreductase is glucose dehydrogenase. 請求項１２または１３に記載の酸化還元酵素・シトクロム複合体を含むバイオセンサ。 A biosensor comprising the oxidoreductase/cytochrome complex according to claim 12 or 13. 請求項１４に記載のバイオセンサに試料を添加し、電位を印加して応答電流を測定し、応答電流に基づいて試料に含まれる測定対象物質の濃度を算出することを特徴とする、測定対象物質の測定方法。 A sample is added to the biosensor according to claim 14, a potential is applied to measure a response current, and a concentration of a substance to be measured contained in the sample is calculated based on the response current. A method of measuring substances. 印加電位が銀・塩化銀電極に対して０～＋３００ｍＶである、請求項１５に記載の測定方法。 The measuring method according to claim 15, wherein the applied potential is 0 to +300 mV with respect to the silver/silver chloride electrode.
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